Screening methods for compounds useful for the treatment of body weight disorders, including obesity

ABSTRACT

The present invention relates to the identification of novel nucleic acid molecules and proteins encoded by such nucleic acid molecules or degenerate variants thereof, that participate in the control of mammalian body weight. The nucleic acid molecules of the present invention represent the genes corresponding to the mammalian tub gene, a gene that is involved in the regulation of body weight.

[0001] This is a continuation of application Ser. No. 09/406,701 filedSep. 24, 1999, which is a divisional of application Ser. No. 09/248,203filed Feb. 10, 1999, now U.S. Pat. No. 6,043,346, which is a divisionalof application no. 08/936,707, filed Sep. 24, 1997, now U.S. Pat. No.5,871,931, which is a divisional of application Ser. No. 08/829,553,filed Mar. 28, 1997, now U.S. Pat. No. 5,817,762, which is a divisionalof application Ser. No. 08/631,200, filed Apr. 12, 1996, now U.S. Pat.No. 5,646,040, which claims priority to provisional application no.60/015,396, filed Apr. 9, 1996, provisional application no. 60/004,424,filed Sep. 28, 1995, provisional application no. 60/002,759, filed Aug.24, 1995, provisional application no. 60/001,444, field Jul. 26, 1995,provisional application no. 60/001,273, filed Jul. 20, 1995, andprovisional application no. 60/000,604, filed Jun. 30, 1995, each ofwhich is incorporated herein by reference in its entirety.

1. INTRODUCTION

[0002] The present invention relates to the mammalian tubby (tub) genes,including the human tub gene, which are novel genes involved in thecontrol of mammalian body weight, including recombinant DNA molecules,cloned genes or degenerate variants thereof. The present inventionfurther relates to novel mammalian, including human, tub gene productsand to antibodies directed against such mammalian tub gene products, orconserved variants or fragments thereof. The present invention alsoincludes cloning vectors containing mammalian tub gene molecules, andhosts which have been transformed with such molecules. In addition, thepresent invention presents methods for the diagnostic evaluation andprognosis of mammalian body weight disorders, including obesity,cachexia and anorexia, and for the identification of subjects exhibitinga predisposition to such conditions. Further, methods and compositionsare presented for the treatment of mammalian body weight disorders,including obesity, cachexia and anorexia. Still further, the presentinvention relates to methods for the use of the mammalian tub geneand/or mammalian tub gene products for the identification of compoundswhich modulate the expression of the mammalian tub gene and/or theactivity of the mammalian tub gene products. Such compounds can be usedas therapeutic agents in the treatment of mammalian body weightdisorders, including obesity, cachexia and anorexia.

2. BACKGROUND OF THE INVENTION

[0003] Obesity represents the most prevalent of body weight disorders,and it is the most important nutritional disorder in the western world,with estimates of its prevalence ranging from 30% to 50% within themiddle-aged population. Other body weight disorders, such as anorexianervosa and bulimia nervosa which together affect approximately 0.2% ofthe female population of the western world, also pose serious healththreats. Further, such disorders as anorexia and cachexia (wasting) arealso prominent features of other diseases such as cancer, cysticfibrosis, and AIDS.

[0004] Obesity, defined as an excess of body fat relative to lean bodymass, also contributes to other diseases. For example, this disorder isresponsible for increased incidences of diseases such as coronary arterydisease, hypertension, stroke, diabetes, hyperlipidemia and somecancers. (See, e.g., Nishina, P. M. et al., 1994, Metab. 43:554-558;Grundy, S. M. & Barnett, J. P., 1990, Dis. Mon. 36:641-731) Obesity isnot merely a behavioral problem, i.e., the result of voluntaryhyperphagia. Rather, the differential body composition observed betweenobese and normal subjects results from differences in both metabolismand neurologic/metabolic interactions. These differences seem to be, tosome extent, due to differences in gene expression, and/or level of geneproducts or activity (Friedman, J. M. et al., 1991, Mammalian Gene1:130-144).

[0005] The epidemiology of obesity strongly shows that the disorderexhibits inherited characteristics (Stunkard, 1990, N. Eng. J. Med.322:1483). Moll et al. have reported that, in many populations, obesityseems to be controlled by a few genetic loci (Moll et al. 1991, Am. J.Hum. Gen. 49:1243). In addition, human twin studies strongly suggest asubstantial genetic basis in the control of body weight, with estimatesof heritability of 80-90% (Simopoulos, A. P. & Childs B., eds., 1989, in“Genetic Variation and Nutrition in Obesity”, World Review of Nutritionand Diabetes 63, S. Karger, Basel, Switzerland; Borjeson, M., 1976,Acta. Paediatr. Scand. 65:279-287).

[0006] Studies of non-obese persons who deliberately attempted to gainweight by systematically over-eating were found to be more resistant tosuch weight gain and able to maintain an elevated weight only by veryhigh caloric intake. In contrast, spontaneously obese individuals areable to maintain their status with normal or only moderately elevatedcaloric intake. In addition, it is a commonplace experience in animalhusbandry that different strains of swine, cattle, etc., have differentpredispositions to obesity. Studies of the genetics of human obesity andof models of animal obesity demonstrate that obesity results fromcomplex defective regulation of both food intake, food induced energyexpenditure and of the balance between lipid and lean body anabolism.

[0007] There are a number of genetic diseases in man and other specieswhich feature obesity among their more prominent symptoms, along with,frequently, dysmorphic features and mental retardation. For example,Prader-Willi syndrome (PWS; reviewed in Knoll, J. H. et al., 1993, Am.J. Med. Genet. 46:2-6) affects approximately 1 in 20,000 live births,and involves poor neonatal muscle tone, facial and genital deformities,and generally obesity.

[0008] In addition to PWS, many other pleiotropic syndromes whichinclude obesity as a symptom have been characterized. These syndromesare more genetically straightforward, and appear to involve autosomalrecessive alleles. The diseases, which include, among others, Ahlstroem,Carpenter, Bardet-Biedl, Cohen, and Morgagni-Stewart-Monel Syndromes.

[0009] A number of models exist for the study of obesity (see, e.g.,Bray, G. A., 1992, Prog. Brain Res. 93:333-341, and Bray, G. A., 1989,Amer. J. Clin. Nutr. 5:891-902). For example, animals having mutationswhich lead to syndromes that include obesity symptoms have also beenidentified. Attempts have been made to utilize such animals as modelsfor the study of obesity, and the best studied animal models, to date,for genetic obesity are mice. For reviews, see e.g., Friedman, J. M. etal., 1991, Mamm. Gen. 1:130-144; Friedman, J. M. and Liebel, R. L.,1992, Cell 69:217-220.)

[0010] Studies utilizing mice have confirmed that obesity is a verycomplex trait with a high degree of heritability. Mutations at a numberof loci have been identified which lead to obese phenotypes. Theseinclude the autosomal recessive mutations obese (ob), diabetes (db), fat(fat) and tubby (tub). In addition, the autosomal dominant mutationsYellow at the agouti locus and Adipose (Ad) have been shown tocontribute to an obese phenotype.

[0011] The ob and db mutations are on chromosomes 6 and 4, respectively,but lead to clinically similar pictures of obesity, evident starting atabout one month of age, which include hyperphagia, severe abnormalitiesin glucose and insulin metabolism, very poor thermoregulation andnon-shivering thermogenesis, and extreme torpor and underdevelopment ofthe lean body mass.

[0012] The ob gene and its human homologue have recently been cloned(Zhang, Y. et al., 1994, Nature 372:425-432). The gene appears toproduce a 4.5 kb adipose tissue messenger RNA which contains a 167 aminoacid open reading frame. The predicted amino acid sequence of the obgene product indicates that it is a secreted protein and may, therefore,play a role as part of a signaling pathway from adipose tissue which mayserve to regulate some aspect of body fat deposition.

[0013] The db locus encodes a high affinity receptor for the ob geneproduct (Chen, H. et al., Cell 84:491-495). The db gene product is asingle membrane-spanning receptor most closely related to the gp130cytokine receptor signal transducing component (Tartaglia, L. A. et al.,1995, Cell 83:1263-1271).

[0014] Homozygous mutations at either the fat or tub loci cause obesitywhich develops more slowly than that observed in ob and db mice(Coleman, D. L., and Eicher, E. M., 1990, J. Heredity 81:424-427), withtub obesity developing slower than that observed in fat animals. Thisfeature of the tub obese phenotype makes the development of tub obesephenotype closest in resemblance to the manner in which obesity developsin humans. Even so, however, the obese phenotype within such animals canbe characterized as massive in that animals eventually attain bodyweights which are nearly two times the average weight seen in normalmice. tub/tub mice develop insulin resistance with their weight gain butdo not progress to overt diabetes.

[0015] In addition to obesity, retinal defects, hearing loss andinfertility have all been observed in tub mice (Heckenlively, 1988, inRetinitis Pigmentosa, Heckenlively, ed., Lippincott, Philadelphia, pp.221-235; Coleman, D. L. & Eicher, E. M., 1990, J. Hered. 81:424-427;Ohlemiller, K. K. et al., 1995, Neuroreport 6:845-849). Several humansyndromes exist in which such defects are found to co-exist with anobesity phenotype, including Bardet-Biedl syndrome, Ahlstroem syndrome,polycystic ovarian disease and Usher's syndrome.

[0016] The fat mutation has been mapped to mouse chromosome 8, while thetub mutation has been mapped to mouse chromosome 7. According to Naggertet al., the fat mutation has recently been identified (Naggert, J. K.,et al., 1995, Nature Genetics 10:135-141). Specifically, the fatmutation appears to be a mutation within the Cpe locus, which encodesthe carboxypeptidase (Cpe) E protein. Cpe is an exopeptidase involved inthe processing of prohormones, including proinsulin.

[0017] The dominant Yellow mutation at the agouti locus, causes apleiotropic syndrome which causes moderate adult onset obesity, a yellowcoat color, and a high incidence of tumor formation (Herberg, L. andColeman, D. L., 1977, Metabolism 26:59), and an abnormal anatomicdistribution of body fat (Coleman, D. L., 1978, Diabetologia14:141-148). This mutation may represent the only known example of apleiotropic mutation that causes an increase, rather than a decrease, inbody size. The mutation causes the widespread expression of a proteinwhich is normally seen only in neonatal skin (Michaud, E. J. et al.,1994, Genes Devel. 8:1463-1472).

[0018] Other animal models include fa/fa (fatty) rats, which bear manysimilarities to the ob/ob and db/db mice, discussed above. Onedifference is that, while fa/fa rats are very sensitive to cold, theircapacity for non-shivering thermogenesis is normal. Torpor seems to playa larger part in the maintenance of obesity in fa/fa rats than in themice mutants. In addition, inbred mouse strains such as NZO mice andJapanese KK mice are moderately obese. Certain hybrid mice, such as theWellesley mouse, become spontaneously fat. Further, several desertrodents, such as the spiny mouse, do not become obese in their naturalhabitats, but do become so when fed on standard laboratory feed.

[0019] Animals which have been used as models for obesity have also beendeveloped via physical or pharmacological methods. For example,bilateral lesions in the ventromedial hypothalamus (VMH) andventrolateral hypothalamus (VLH) in the rat are associated,respectively, with hyperphagia and gross obesity and with aphagia,cachexia and anorexia. Further, it has been demonstrated that feedingmonosodium-glutamate (MSG) or gold thioglucose to newborn mice alsoresults in an obesity syndrome.

[0020] In summary, therefore, obesity, which poses a major, worldwidehealth problem, represents a complex, highly heritable trait. Given theseverity, prevalence and potential heterogeneity of such disorders,there exists a great need for the identification of those genes thatparticipate in the control of body weight.

[0021] It is an objective of the invention to provide a modulators, suchas intracellular modulators, of body weight, to provide methods fordiagnosis of body weight disorders, to provide therapy for suchdisorders and to provide an assay system for the screening of substanceswhich can be used to control body weight.

3. SUMMARY OF THE INVENTION

[0022] The present invention relates to the identification of novelnucleic acid molecules and proteins encoded by such nucleic acidmolecules or degenerate variants thereof, that participate in thecontrol of mammalian body weight. The nucleic acid molecules of thepresent invention represent the genes corresponding to the mammalian tubgene, including the human tub gene, which are involved in theregulation, control and/or modulation of body weight.

[0023] In particular, the compositions of the present invention includenucleic acid molecules (e.g., tub gene), including recombinant DNAmolecules, cloned genes or degenerate variants thereof, especiallynaturally occurring variants, which encode novel tub gene products, andantibodies directed against such tub gene products or conserved variantsor fragments thereof. The compositions of the present inventionadditionally include cloning vectors, including expression vectors,containing the nucleic acid molecules of the invention and hosts whichhave been transformed with such nucleic acid molecules.

[0024] Nucleic acid sequences of a wild type and a mutant form of themurine tub gene are provided. The wild type murine tub gene produces afull length transcript of approximately 7.0 kb and encodes a protein of505 amino acids, the sequence of which is provided. The amino acidsequence of the predicted full length tub gene product does not containeither a recognizable transmembrane domain or a signal sequence,suggesting that the tub gene product is an intracellular gene product.The mammalian tub gene is, as shown herein, expressed in the brain,including the hypothalamus.

[0025] Nucleic acid sequences of a wild type human tub gene are alsoprovided. The human tub gene encodes a full length protein of 505 aminoacids, the sequence of which is provided. The human tub gene and geneproduct are strikingly similar to the murine tub gene and gene product.Specifically, the human tub gene is, at the nucleotide level, 89%identical to the murine tub gene. Further, the amino acid sequence ofthe human tub gene product is 94% identical to the amino acid sequenceof the murine tub gene product.

[0026] Both murine and human tub genes produce transcripts which undergoalternative splicing. Such alternative splicing yields, in addition tothe full length transcripts, transcripts which lack sequencescorresponding to tub exon 5. Nucleic acid sequences corresponding tosuch alternatively spliced transcripts and the tub gene products encodedby such alternatively spliced transcripts are provided herein.

[0027] In addition, this invention presents methods for the diagnosticevaluation and prognosis of body weight disorders, including obesity,cachexia and anorexia, and for the identification of subjects having apredisposition to such conditions. For example, nucleic acid moleculesof the invention can be used as diagnostic hybridization probes or asprimers for diagnostic PCR analysis for the identification of tub genemutations, allelic variations and regulatory defects in the tub gene,and of alternatively spliced transcripts produced by the tub gene. Forexample, human tub genomic sequences are provided which can be used toselectively amplify human tub exons for analysis.

[0028] Further, methods and compositions are presented for the treatmentof body weight disorders, including obesity, cachexia and anorexia. Suchmethods and compositions are capable of modulating the level of tub geneexpression and/or the level of tub gene product activity. Such methodsand compositions can also be utilized in the treatment or ameliorationof symptoms of tub gene-related sensory defects (e.g., eye and hearing)and fertility defects.

[0029] Still further, the present invention relates to methods for theuse of the tub gene and/or tub gene products for the identification ofcompounds which modulate tub gene expression and/or the activity of tubgene products. Such compounds can be used as agents to control bodyweight and, in particular, therapeutic agents in the treatment of bodyweight and body weight disorders, including obesity, cachexia andanorexia. Such methods and compositions can also be utilized in thetreatment or amelioration of symptoms of tub gene-related sensory (e.g.,eye and hearing) and fertility defects. It is further contemplated thatthe nucleic acid molecules, peptides and other compounds of theinvention can have agricultural applications. For example, the ratio offat to lean tissue of agricultural animals can be favorably altered,e.g., this ratio can be decreased.

[0030] This invention is based, in part, on the genetic and physicalmapping of the tub gene to a specific portion of mouse chromosome 7,described in the Examples presented, below, in Section 6 and 7. Theinvention is further based, in part, on the expression and sequenceanalysis of a candidate tub homozygous animals, which proves that thiscandidate gene does, indeed, represent the tub gene. Such analyses aredescribed in the Examples presented, below, in Sections 8-12, andinclude the identification of a splice site mutation in nucleic acidderived from tub animals which is absent from the corresponding nucleicacid derived from wild type, non-obese animals. This single basemutation consists of a guanine (G) to a thymidine (T) in the splice siterecognition sequence, which results in the retention of an intronicsequence in the mature tub mRNA that encodes an abnormal,loss-of-function, tub gene product. Further Section 13 presents thesuccessful cloning of the human tub gene homologue.

[0031] Still further, the Example presented in Section 14 demonstratesthat both the murine and human tub transcripts undergo alternativesplicing. Section 15 demonstrates the successful expression ofrecombinant human and murine tub gene products. The Example presented inSection 16 describes the identification, cloning and characterization ofa human tub homolog.

4. DESCRIPTION OF THE FIGURES

[0032]FIG. 1. Physical map of the D7Mit17 to D7Mit53 interval of mousechromosome 7.

[0033]FIG. 2. Northern blot analysis of total RNA derived from varioustissues of tub and wild type (C57BL/6J) mice, using the 90 bp P8X1 DNAfragment as a probe. See Sections 10.1 and 10.2 for details.

[0034]FIG. 3. Northern blot analysis of total RNA derived from varioustissues of tub and wild type (C57BL/6J) mice, using the 1.15 kb fume009cDNA clone as a probe. See Sections 9.1 and 9.2 for details.

[0035]FIG. 4. Southern blot analysis of EcoRI-digested mammalian genomicDNA derived from a number of different species, as indicated, using afragment of CBT9 (P8X9-10) as a probe, as described, below, in Sections10.1 and 10.2.

[0036]FIG. 5. In situ hybridization analysis of CBT9 spatial expressionin a brain (hypothalamus) tissue section of C57BL/6J wild type mice,using a fume009 cDNA probe.

[0037]FIG. 6A-6D. Nucleotide sequence of the coding region (and portionsof 5′ and 3′ untranslated regions) of the wild type tub gene (bottomline) (SEQ ID NO:1) and the encoded amino acid sequence (top line) (SEQID NO:2).

[0038]FIG. 7A-7D. Alignment of cDNA and genomic sequences derived fromwild type C57BL/6J (genomic=SEQ ID NO:4; cDNA=SEQ ID NO:6) and tub RNA(genomic=SEQ ID NO:3; cDNA=SEQ ID NO:5) in the region of the splice sitemutation. See Section 12.1 and 12.2 for details.

[0039]FIG. 8. Schematic representation of the splicing defect in theCBT9 gene in tub animals.

[0040]FIG. 9A-9D. Nucleotide sequence of the coding region (and portionsof 5′ and 3′ untranslated regions) of the human tub gene (bottom line)(SEQ ID NO:7) and the encoded human tub gene product amino acid sequence(top line) (SEQ ID NO:8).

[0041]FIG. 10A-10G. Human tub genomic sequence. Depicted herein arehuman tub gene exons 4-12 nucleotide sequences and flanking intronicsequences. Intron boundaries are depicted in bold; exon sequences areunderlined. 10A. (SEQ ID NO:9) Exon 4 (corresponding to nucleotidesequence 254-397 of FIG. 9) and its flanking genomic sequence. 10B. (SEQID NO:10) Exon 5 (corresponding to nucleotide sequence 398-565 of FIG.9) and its flanking genomic sequence. 10C-10D. (SEQ ID NO:11 Exons 6-8(corresponding to nucleotide sequences 566-687, 688-885, and 886-998 ofFIG. 9A-9D, respectively) and its flanking genomic sequence. 10E. (SEQID NO:12) Exon 9 (corresponding to nucleotide sequence 999-1116 of FIG.9) and its flanking genomic sequence. 10F-10G. (SEQ ID NO:13) Exons10-12 (corresponding to nucleotide sequences 1117-1215, 1216-1387 and1388-1729 of FIG. 9A-9D, respectively) and its flanking genomicsequence.

[0042]FIG. 11. SDS polyacrylamide protein gel demonstrating bacterialexpression of recombinant murine and human tub gene products. Lanes fromleft to right: Pharmacia Low Molecular Weight Markers; uninducedBL21DE3/human pET29*-tub; induced BL21DE3/human pET29*-tub; inducedBL21DE3/ human pET29*-tub HIS₆; induced BL21DE3/murine pET29*-tub;induced BL21DE3/murine pET29*-tub HIS₆. Arrow represents recombinant tubgene products.

[0043]FIG. 12A-12C. Nucleotide and amino acid sequence of the human tubhomolog 1 gene. Top line: amino acid sequence. (SEQ ID NO:15) Bottomline: nucleotide sequence. (SEQ ID NO:14) “*” represents the stop codon.

5. DETAILED DESCRIPTION OF THE INVENTION

[0044] Described herein are the identification of the novel mammaliantubby (tub) genes, including the human tub gene, which are involved inthe control of mammalian body weight. Also described are recombinantmammalian, including human, tub DNA molecules, cloned genes, ordegenerate variants thereof. The compositions of the present inventionfurther include tub gene products (e.g., proteins) that are encoded bythe tub gene, and the modulation of tub gene expression and/or tub geneproduct activity in the treatment of mammalian body weight, and bodyweight disorders, including obesity, cachexia and anorexia. Alsodescribed herein are antibodies against tub gene products (e.g.,proteins), or conserved variants or fragments thereof, and nucleic acidprobes useful for the identification of tub gene mutations and the useof such nucleic acid probes in diagnosing mammalian body weightdisorders, including obesity, cachexia and anorexia. Further describedare methods for the use of the tub gene and/or tub gene products in theidentification of compounds which modulate the activity of the tub geneproduct.

[0045] The murine tub nucleic acid compositions of the invention aredemonstrated in the Examples presented, below, in Sections 6 through 12.The human tub nucleic acid compositions of the invention aredemonstrated in Section 13, below. For clarity, it should be noted thatthe murine tub gene is also referred to herein as the CBT9 gene, and wasidentified and cloned as follows. Genetic and physical mapping of themurine tub gene interval was narrowed to the interval between markersD7Mit39 and D7Mit53. A P1 genomic clone, P8, was located within thisinterval, as indicated in FIG. 1. A P8 subclone, designated ium008p004,was sequenced. An analysis of ium008p004 indicated that this sequencewas part of the coding region of a gene. A 90 bp fragment, designatedP8X1, was amplified from this ium008p004 subclone. P8X1 was used as aprobe to screen a mouse brain cDNA library, resulting in theidentification of a 1.15 kb cDNA clone, designated fume009. fume009 wasused as a probe to screen a mouse hypothalamus cDNA library, resultingin the identification of a 6.0 kb cDNA clone, designated fumhol9. Tosummarize, therefore, ium008p004, PX81, fume009 and fumh019 are all partof the murine tub gene, which is also referred to herein as the CBT9gene.

5.1. The Tub Gene

[0046] The murine tub gene, shown in FIG. 6A-6D, and the human tub gene,shown in FIG. 9A-9D, are novel genes involved in the control of bodyweight. Nucleic acid sequences of the identified tub gene are describedherein. As used herein, “tub gene” refers to (a) a gene containing theDNA sequence shown in FIG. 6A-6D or FIG. 9A-9D or contained in the cDNAclone fumh019, CBT9H1 or CBT9H3, or genomic clone P6, P8, or B13, asdeposited with the American Type Culture Collection (ATCC); (b) any DNAsequence that encodes the amino acid sequence shown in FIG. 6A-6D orFIG. 9A-9D, or encodes the amino acid sequence shown in FIG. 6A-6D orFIG. 9A-9D but lacking the amino acid residues encoded by tub exon 5(i.e., amino acid residues 134-189 to 134-189 of FIG. 6A-6D or FIG.9A-9D), or encoded by the cDNA clone fumh019, CBT9H1 or CBT9H3, orgenomic clone P6, P8, or B13, as deposited with the ATCC; (c) any DNAsequence that hybridizes to the complement of the DNA sequences thatencode the amino acid sequence shown in FIG. 6A-6D or FIG. 9A-9D, orencodes the amino acid sequence shown in FIG. 6A-6D or FIG. 9A-9D butlacking the amino acid residues encoded by tub exon 5 (i.e., amino acidresidues 134 to 189 of FIG. 6A-6D or FIG. 9A-9D), or contained in thecDNA clone fumh019, CBT9H1 or CBT9H3, or genomic clone P6, P8, or B13,as de-posited with the ATCC, under highly stringent conditions, e.g.,hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecylsulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at68° C. (Ausubel F. M. et al., eds., 1989, Current Protocols in MolecularBiology, Vol. I, Green Publishing Associates, Inc., and John Wiley &sons, Inc., New York, at p. 2.10.3) and encodes a gene productfunctionally equivalent to a tub gene product encoded by sequencescontained within the cDNA clone fumh019, CBT9H1 or CBT9H3, sequencesshown in FIG. 6A-6D or FIG. 9A-9D, sequences shown in FIG. 6A-6D or FIG.9A-9D, but lacking tub exon 5, or genomic clone P6, P8, or B13; and/or(d) any DNA sequence that hybridizes to the complement of the DNAsequences that encode the amino acid sequence shown in FIG. 6A-6D orFIG. 9A-9D, or encode the amino acid sequence shown in FIG. 6A-6D orFIG. 9A-9D but lacking the amino acid residues encoded by tub exon 5(i.e., amino acid residues 134 to 189 of FIG. 6A-6D or FIG. 9A-9D),contained in the cDNA clone fumh019, CBT9H1 or CBT9H3, or genomic cloneP6, P8, or B13, as deposited with the ATCC, under less stringentconditions, such as moderately stringent conditions, e.g., washing in0.2×SSC/0.1% SDS at 42° C. (Ausubel et al., 1989, supra), yet whichstill encodes a functionally equivalent tub gene product. As usedherein, tub gene may also refer to degenerate variants of DNA sequences(a) through (d), especially naturally occurring variants thereof.

[0047] The invention also includes nucleic acid molecules, preferablyDNA molecules, that hybridize to, and are therefore the complements of,the DNA sequences (a) through (d), in the preceding paragraph. Suchhybridization conditions may be highly stringent or less highlystringent, as described above. In instances wherein the nucleic acidmolecules are deoxyoligonucleotides (“oligos”), highly stringentconditions may refer, e.g., to washing in 6×SSC/0.05% sodiumpyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-baseoligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).These nucleic acid molecules may encode or act as tub gene antisensemolecules, useful, for example, in tub gene regulation (for and/or asantisense primers in amplification reactions of tub gene nucleic acidsequences. With respect to tub gene regulation, such techniques can beused to regulate, for example, cachexia and/or anorexia. Further, suchsequences may be used as part of ribozyme and/or triple helix sequences,also useful for tub gene regulation. Still further, such molecules maybe used as components of diagnostic methods whereby, for example, thepresence of a particular tub allele or alternatively spliced tubtranscript responsible for causing or predisposing one to a weightdisorder, such as obesity, may be detected. Among the molecules whichcan be used for diagnostic methods such as these which involveamplification of genomic tub sequences are those listed in FIG. 10A-10Gand in Table I, below.

[0048] The invention also encompasses (a) DNA vectors that contain anyof the foregoing tub coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingtub coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences; and (c) geneticallyengineered host cells that contain any of the foregoing tub codingsequences operatively associated with a regulatory element that directsthe expression of the coding sequences in the host cell. As used herein,regulatory elements include but are not limited to inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include but are not limited to the cytomegalovirushCMV immediate early gene, the early or late promoters of SV40adenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage A, the controlregions of fd coat protein, the promoter for 3-phosphoglycerate kinase,the promoters of acid phosphatase, and the promoters of the yeastα-mating factors. The invention includes fragments of any of the DNAsequences disclosed herein.

[0049] In addition to the tub gene sequences described above, homologsof such sequences, exhibiting extensive homology to one or more ofdomains of the tub gene product present in other species can beidentified and readily isolated, without undue experimentation, bymolecular biological techniques well known in the art. Further, therecan exist homolog genes at other genetic loci within the genome thatencode proteins which have extensive homology to one or more domains ofthe tub gene product. These genes can also be identified via similartechniques.

[0050] As an example, in order to clone a human tub gene homologue usingisolated murine tub gene sequences as disclosed herein, such murine tubgene sequences may be labeled and used to screen a cDNA libraryconstructed from mRNA obtained from appropriate cells or tissues (e.g.,preferably hypothalamus, or brain) derived from the organism (in thiscase, human) of interest. With respect to the cloning of such a humantub homologue, a human fetal brain cDNA library (e.g., Clontech#HL1149x) may, for example, be used for screening.

[0051] The hybridization washing conditions used should be of a lowerstringency when the cDNA library is derived from an organism differentfrom the type of organism from which the labeled sequence was derived.With respect to the cloning of a human tub homologue, for example,hybridization can be performed for 4 hours at 65° C. using AmershamRapid Hyb™ buffer (Cat. #RPN1639) according to manufacturer's protocol,followed by washing, with a final washing stringency of 1.0×SSC/0.1% SDSat 50° C. for 20 minutes being preferred.

[0052] Low stringency conditions are well known to those of skill in theart, and will vary predictably depending on the specific organisms fromwhich the library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y.;and Ausubel et al., 1989, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y.

[0053] Alternatively, the labeled fragment may be used to screen agenomic library derived from the organism of interest, again, usingappropriately stringent conditions.

[0054] Further, a tub gene homologue may be isolated from nucleic acidof the organism of interest by performing PCR using two degenerateoligonucleotide primer pools designed on the basis of amino acidsequences within the tub gene product disclosed herein. The template forthe reaction may be cDNA obtained by reverse transcription of mRNAprepared from, for example, human or non-human cell lines or tissueknown or suspected to express a tub gene allele.

[0055] The PCR product may be subcloned and sequenced to ensure that theamplified sequences represent the sequences of a tub gene nucleic acidsequence. The PCR fragment may then be used to isolate a full lengthcDNA clone by a variety of methods. For example, the amplified fragmentmay be labeled and used to screen a cDNA library, such as abacteriophage cDNA library. Alternatively, the labeled fragment may beused to isolate genomic clones via the screening of a genomic library.

[0056] Taking, as an example, the cloning of a human tub homologue usingmurine tub nucleic acid sequences, among the murine tub primers whichmay be utilized for PCR amplification are, for example, the following,which are derived from the murine fumh019 sequence described, above:

[0057] 5′-CCG ACT CGA TTG CCA GTG TA-3′ (SEQ ID NO:16)

[0058] 5′-GCG GAT ACA GAC TCT CTC AT-3′ (SEQ ID NO:17)

[0059] These primers generate a cDNA product of approximately 950 basepairs which can then be used as probe for the screening of appropriatecDNA libraries such as, for example, human fetal brain cDNA libraries(e.g., Clontech #HL1149x). When a cDNA library is screened with probessuch as this, hybridization can, for example, be performed for 4 hoursat 65° C. using Amersham Rapid Hyb™ buffer (Cat. #RPN1639) according tomanufacturer's protocol, followed by washing, with a final washingstringency of 1.0×SSC/0.1% SDS at 50° C. for 20 minutes being preferred.

[0060] The Example presented in Section 16, below, describes thesuccessful identification, cloning and characterization of a human tubhomolog.

[0061] PCR technology may also be utilized to isolate full length cDNAsequences. For example, RNA may be isolated, following standardprocedures, from an appropriate cellular or tissue source (i.e., oneknown, or suspected, to express the tub gene, such as, for example,hypothalamus tissue). A reverse transcription reaction may be performedon the RNA using an oligonucleotide primer specific for the most 5′ endof the amplified fragment for the priming of first strand synthesis. Theresulting RNA/DNA hybrid may then be “tailed” with guanines using astandard terminal transferase reaction, the hybrid may be digested withRNAase H, and second strand synthesis may then be primed with a poly-Cprimer. Thus, cDNA sequences upstream of the amplified fragment mayeasily be isolated. For a review of cloning strategies which may beused, see e.g., Sambrook et al., 1989, supra.

[0062] tub gene sequences may additionally be used to isolate mutant tubgene alleles. Such mutant alleles may be isolated from individualseither known or proposed to have a genotype which contributes to thesymptoms of body weight disorders such as obesity, cachexia or anorexia.Mutant alleles and mutant allele products may then be utilized in thetherapeutic and diagnostic systems described below. Additionally, suchtub gene sequences can be used to detect tub gene regulatory (e.g.,promoter) defects which can affect body weight.

[0063] A cDNA of a mutant tub gene may be isolated, for example, byusing PCR, a technique which is well known to those of skill in the art.In this case, the first cDNA strand may be synthesized by hybridizing anoligo-dT oligonucleotide to mRNA isolated from tissue known or suspectedto be expressed in an individual putatively carrying the mutant tuballele, and by extending the new strand with reverse transcriptase. Thesecond strand of the cDNA is then synthesized using an oligonucleotidethat hybridizes specifically to the 5′ end of the normal gene. Usingthese two primers, the product is then amplified via PCR, cloned into asuitable vector, and subjected to DNA sequence analysis through methodswell known to those of skill in the art. By comparing the DNA sequenceof the mutant tub allele to that of the normal tub allele, themutation(s) responsible for the loss or alteration of function of themutant tub gene product can be ascertained.

[0064] Alternatively, a genomic library can be constructed using DNAobtained from an individual suspected of or known to carry the mutanttub allele, or a cDNA library can be constructed using RNA from a tissueknown, or suspected, to express the mutant tub allele. The normal tubgene or any suitable fragment thereof may then be labeled and used as aprobe to identify the corresponding mutant tub allele in such libraries.Clones containing the mutant tub gene sequences may then be purified andsubjected to sequence analysis according to methods well known to thoseof skill in the art.

[0065] Additionally, an expression library can be constructed utilizingcDNA synthesized from, for example, RNA isolated from a tissue known, orsuspected, to express a mutant tub allele in an individual suspected ofor known to carry such a mutant allele. In this manner, gene productsmade by the putatively mutant tissue may be expressed and screened usingstandard antibody screening techniques in conjunction with antibodiesraised against the normal tub gene product, as described, below, inSection 5.3. (For screening techniques, see, for example, Harlow, E. andLane, eds., 1988, “Antibodies: A Laboratory Manual”, Cold Spring HarborPress, Cold Spring Harbor.) In cases where a tub mutation results in anexpressed gene product with altered function (e.g., as a result of amissense or a frameshift mutation), a polyclonal set of anti-tub geneproduct antibodies are likely to cross-react with the mutant tub geneproduct. Library clones de-tected via their reaction with such labeledantibodies can be purified and subjected to sequence analysis accordingto methods well known to those of skill in the art.

5.2. Protein Products of the Tub Gene

[0066] tub gene products, or peptide fragments thereof, can be preparedfor a variety of uses. For example, such gene products, or peptidefragments thereof, can be used for the generation of antibodies, indiagnostic assays, or for the identification of other cellular geneproducts involved in the regulation of body weight.

[0067] The amino acid sequence depicted in FIG. 6A-6D represents amurine tub gene product, while the amino acid se-quence depicted in FIG.9A-9D represents a human tub gene product. The tub gene product,sometimes referred to herein as a “tub protein”, may additionallyinclude those gene products encoded by the tub gene sequences describedin Section 5.1, above, and is intended to include, for example, a tubgene product encoded by a tub gene sequence lacking tub exon 5.

[0068] In addition, tub gene products may include proteins thatrepresent functionally equivalent gene products. Such an equivalent tubgene product may contain deletions, additions or substitutions of aminoacid residues within the amino acid sequence encoded by the tub genesequence described, above, in Section 5.1, but which result in a silentchange, thus producing a functionally equivalent tub gene product. Aminoacid substitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

[0069] “Functionally equivalent”, as utilized herein, refers to aprotein capable of exhibiting a substantially similar in vivo activityas the endogenous tub gene products encoded by the tub gene sequencesdescribed in Section 5.1, above. The in vivo activity of the tub geneproduct, as used herein, refers to the ability of the tub gene product,when present in an appropriate cell type, to ameliorate, prevent ordelay the appearance of the obese phenotype relative to it appearancewhen that cell type lacks a functional tub gene product. “Obesephenotype”, as used herein, refers to the well known tub phenotype, dbphenotype, or ob phenotype. In humans, this can also refer to anincreased percentage of body fat which is medically considered abnormal.

[0070] The tub gene products or peptide fragments thereof, may beproduced by recombinant DNA technology using techniques well known inthe art. Thus, methods for preparing the tub gene polypeptides andpeptides of the invention by expressing nucleic acid containing tub genesequences are described herein. Methods which are well known to thoseskilled in the art can be used to construct expression vectorscontaining tub gene product coding sequences and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. See, for example, thetechniques described in Sambrook et al., 1989, supra, and Ausubel etal., 1989, supra. Alternatively, RNA capable of encoding tub geneproduct sequences may be chemically synthesized using, for example,synthesizers. See, for example, the techniques described in“Oligonucleotide Synthesis”, 1984, Gait, M. J. ed., IRL Press, Oxford,which is incorporated by reference herein in its entirety.

[0071] A variety of host-expression vector systems may be utilized toexpress the tub gene coding sequences of the invention. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, exhibit the tub gene product ofthe invention in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing tub gene product coding sequences; yeast (e.g.,Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing the tub gene product coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing the tub gene product coding sequences; plantcell systems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing tub gene product coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).

[0072] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the tub geneproduct being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of tub protein or for raising antibodies to tub protein,for example, vectors which direct the expression of high levels offusion protein products that are readily purified may be desirable. Suchvectors include, but are not limited, to the E. coli expression vectorpUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the tub geneproduct coding sequence may be ligated individually into the vector inframe with the lac Z coding region so that a fusion protein is produced;pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; VanHeeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like.pGEX vectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetgene product can be released from the GST moiety. The Example presentedin Section 15, below, describes the successful expression of both murineand human recombinant tub gene products utilizing modified pET vectors(Novagen, Inc., Madison Wis.).

[0073] In an insect system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The tub gene coding sequence maybe cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter). Successful insertion oftub gene coding sequence will result in inactivation of the polyhedringene and production of non-occluded recombinant virus (i.e., viruslacking the proteinaceous coat coded for by the polyhedrin gene). Theserecombinant viruses are then used to infect Spodoptera frugiperda cellsin which the inserted gene is expressed. (E.g., see Smith et al., 1983,J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).

[0074] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the tub gene coding sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing tub gene product in infected hosts.(Eq., See Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659).Specific initiation signals may also be required for efficienttranslation of inserted tub gene product coding sequences. These signalsinclude the ATG initiation codon and adjacent sequences. In cases wherean entire tub gene, including its own initiation codon and adjacentsequences, is inserted into the appropriate expression vector, noadditional translational control signals may be needed. However, incases where only a portion of the tub gene coding sequence is inserted,exogenous translational control signals, including, perhaps, the ATGinitiation codon, must be provided. Furthermore, the initiation codonmust be in phase with the reading frame of the desired coding sequenceto ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., 1987,Methods in Enzymol. 153:516-544).

[0075] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK,293, 3T3, WI38, and in particular, hypothalamic cell lines such as GNand GH-1 cell lines.

[0076] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the tub gene product may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the tub geneproduct. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of the tub gene product.

[0077] The Example presented in Section 15, below, describes thesuccessful expression of recombinant tub gene products in mammalian celllines.

[0078] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler, et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), andadenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817)genes can be employed in tk⁻, hgprt⁻or aprt⁻cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147).

[0079] Alternatively, any fusion protein may be readily purified byutilizing an antibody specific for the fusion protein being expressed.For example, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system, the gene of interest is subcloned into avaccinia recombination plasmid such that the gene's open reading frameis translationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni²⁺.nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers. The Example presented in Section 15,below, demonstrates the successful expression of carboxy-terminalhistidine-tagged recombinant tub gene products.

[0080] The tub gene products can also be expressed in transgenicanimals. Animals of any species, including, but not limited to, mice,rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-humanprimates, e g., baboons, monkeys, and chimpanzees may be used togenerate tub transgenic animals.

[0081] Any technique known in the art may be used to introduce the tubgene transgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); genetargeting in embryonic stem cells (Thompson et al., 1989, Cell56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57:717-723); etc. For a review of such techniques, see Gordon,1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which isincorporated by reference herein in its entirety.

[0082] The present invention provides for transgenic animals that carrythe tub transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals. Thetransgene may be integrated as a single transgene or in concatamers,e.g., head-to-head tandems or head-to-tail tandems. The transgene mayalso be selectively introduced into and activated in a particular celltype by following, for example, the teaching of Lasko et al. (Lasko, M.et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6232-6236). The regulatorysequences required for such a cell-type specific activation will dependupon the particular cell type of interest, and will be apparent to thoseof skill in the art. When it is desired that the tub gene transgene beintegrated into the chromosomal site of the endogenous tub gene, genetargeting is preferred. Briefly, when such a technique is to beutilized, vectors containing some nucleotide sequences homologous to theendogenous tub gene are designed for the purpose of integrating, viahomologous recombination with chromosomal sequences, into and disruptingthe function of the nucleotide sequence of the endogenous tub gene. Thetransgene may also be selectively introduced into a particular celltype, thus inactivating the endogenous tub gene in only that cell type,by following, for example, the teaching of Gu et al. (Gu, et al., 1994,Science 265: 103-106). The regulatory sequences required for such acell-type specific inactivation will depend upon the particular celltype of interest, and will be apparent to those of skill in the art.

[0083] Once transgenic animals have been generated, the expression ofthe recombinant tub gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include but are not limited to Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of tub gene-expressing tissue, may also beevaluated immunocytochemically using antibodies specific for the tubtransgene product.

5.3. Antibodies to the Gene Products

[0084] Described herein are methods for the production of antibodiescapable of specifically recognizing one or more tub gene productepitopes or epitopes of conserved variants or peptide fragments of thetub gene products.

[0085] Such antibodies may include, but are not limited to, polyclonalantibodies, monoclonal antibodies (mAbs), humanized or chimericantibodies, single chain antibodies, Fab fragments, F(ab′)₂ fragments,fragments produced by a Fab expression library, anti-idiotypic (anti-Id)antibodies, and epitope-binding fragments of any of the above. Suchantibodies may be used, for example, in the detection of a tub geneproduct in an biological sample and may, therefore, be utilized as partof a diagnostic or prognostic technique whereby patients may be testedfor abnormal levels of tub gene products, and/or for the presence ofabnormal forms of the such gene products. Such antibodies may also beutilized in conjunction with, for example, compound screening schemes,as described, below, in Section 5.4.2, for the evaluation of the effectof test compounds on tub gene product levels and/or activity.Additionally, such antibodies can be used in conjunction with the genetherapy techniques described, below, in Section 5.4.3, to, for example,evaluate the normal and/or engineered tub-expressing cells prior totheir introduction into the patient.

[0086] Anti-tub gene product antibodies may additionally be used as amethod for the inhibition of abnormal tub gene product activity. Thus,such antibodies may, therefore, be utilized as part of weight disordertreatment methods.

[0087] For the production of antibodies against a tub gene product,various host animals may be immunized by injection with a tub geneproduct, or a portion thereof. Such host animals may include but are notlimited to rabbits, mice, and rats, to name but a few. Various adjuvantsmay be used to increase the immunological response, depending on thehost species, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum.

[0088] Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as a tub gene product, or an antigenic functional derivativethereof. For the production of polyclonal antibodies, host animals suchas those described above, may be immunized by injection with tub geneproduct supplemented with adjuvants as also described above.

[0089] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, may be obtained by any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497;and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc.Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique(Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulinclass including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Thehybridoma producing the mAb of this invention may be cultivated in vitroor in vivo. Production of high titers of mAbs in vivo makes this thepresently preferred method of production.

[0090] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion.

[0091] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 334:544-546) can be adaptedto produce single chain antibodies against tub gene products. Singlechain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

[0092] Antibody fragments which recognize specific epitopes may begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.

5.4. Uses of the Tub Gene, Gene Products, and Antibodies

[0093] Described herein are various applications of the tub gene, thetub gene product including peptide fragments thereof, and of antibodiesdirected against the tub gene product and peptide fragments thereof.Such applications include, for example, prognostic and diagnosticevaluation of body weight disorders and the identification of subjectswith a predisposition to such disorders, as described, below, in Section5.4.1. Additionally, such applications include methods for the treatmentof body weight and body weight disorders, as described, below, inSection 5.4.2, and for the identification of compounds which modulatethe expression of the tub gene and/or the activity of the tub geneproduct, as described below, in Section 5.4.3. Such compounds caninclude, for example, other cellular products which are involved in bodyweight regulation. These compounds can be used, for example, in theamelioration of body weight disorders including obesity, cachexia andanorexia.

[0094] While, for clarity, uses related to body weight disorderabnormalities are primarily described in this Section, it is to be notedthat each of the diagnostic and therapeutic treatments described hereincan additionally be utilized in connection with sensory (e.g., eye andhearing) and fertility defects that are commonly associated withmutations in the tub gene. That is, the diagnostic and prognostictechniques described herein can also be utilized to diagnose tub relatedeye, hearing and fertility abnormalities and/or a predisposition towardthe development of such eye, hearing and fertility abnormalities, whilethe therapeutic techniques described herein can be utilized for theamelioration of such eye, hearing and fertility defects.

5.4.1. Diagnosis of Body Weight Disorder Abnormalities

[0095] A variety of methods can be employed for the diagnostic andprognostic evaluation of body weight disorders, including obesity,cachexia and anorexia, and for the identification of subjects having apredisposition to such disorders.

[0096] Such methods may, for example, utilize reagents such as the tubgene nucleotide sequences described in Sections 5.1, and antibodiesdirected against tub gene products, including peptide fragments thereof,as described, above, in Section 5.3. Specifically, such reagents may beused, for example, for: (1) the detection of the presence of tub genemutations, or the detection of either over- or under-expression of tubgene mRNA relative to the non-body weight disorder state or thequalitative or quantitative detection of alternatively spliced forms oftub transcripts which may correlate with certain body weight disordersor susceptibility toward such body weight disorders; and (2) thedetection of either an over- or an under-abundance of tub gene productrelative to the non-body weight disorder state or the presence of amodified (e.g., less than full length) tub gene product which correlateswith a body weight disorder state or a progression toward a body weightdisorder state.

[0097] The methods described herein may be performed, for example, byutilizing pre-packaged diagnostic kits comprising at least one specifictub gene nucleic acid or anti-tub gene antibody reagent describedherein, which may be conveniently used, e.g., in clinical settings, toscreen and diagnose patients exhibiting body weight disorderabnormalities and to screen and identify those individuals exhibiting apredisposition to developing a body weight disorder abnormality.

[0098] For the detection of tub mutations, any nucleated cell can beused as a starting source for genomic nucleic acid. For the detection oftub transcripts or tub gene products, any cell type or tissue in whichthe tub gene is expressed, such as, for example, hypothalamus cells, maybe utilized.

[0099] Nucleic acid-based detection techniques are described, below, inSection 5.4.1.1. Peptide detection techniques are described, below, inSection 5.4.1.2.

5.4.1.1. Detection of Tub Gene Nucleic Acid Molecules

[0100] Mutations or polymorhisms within the tub gene can be detected byutilizing a number of techniques. Nucleic acid from any nucleated cellcan be used as the starting point for such assay techniques, and may beisolated according to standard nucleic acid preparation procedures whichare well known to those of skill in the art.

[0101] Genomic DNA may be used in hybridization or amplification assaysof biological samples to detect abnormalities involving tub genestructure, including point mutations, insertions, deletions andchromosomal rearrangements. Such assays may include, but are not limitedto, direct sequencing (Wong, C. et al., 1987, Nature 330:384-386),single stranded conformational polymorphism analyses (SSCP; Orita, M. etal., 1989, Proc. Natl. Acad. Sci. USA 86:2766-2770), heteroduplexanalysis (Keen, T. J. et al., 1991, Genomics 11:199-205; Perry, D. J. &Carrell, R. W., 1992), denaturing gradient gel electrophoresis (DGGE;Myers, R. M. et al., 1985, Nucl. Acids Res. 13:3131-3145), chemicalmismatch cleavage (Cotton, R. G. et al., 1988, Proc. Natl. Acad. Sci.USA 85:4397-4401) and oligonucleotide hybridization (Wallace, R. B. etal., 1981, Nucl. Acids Res. 9:879-894; Lipshutz, R. J. et al., 1995,Biotechniques 19:442-447).

[0102] Diagnostic methods for the detection of tub gene specific nucleicacid molecules, in patient samples or other appropriate cell sources,may involve the amplification of specific gene sequences, e.g., by thepolymerase chain reaction (PCR; the experimental embodiment set forth inMullis, K. B., 1987, U.S. Pat. No. 4,683,202), followed by the analysisof the amplified molecules using techniques well known to those of skillin the art, such as, for example, those listed above. Utilizing analysistechniques such as these, the amplified sequences can be compared tothose which would be expected if the nucleic acid being amplifiedcontained only normal copies of the tub gene in order to determinewhether a tub gene mutation exists.

[0103] Among those tub nucleic acid sequences which are preferred forsuch amplification-related diagnostic screening analyses areoligonucleotide primers which amplify tub exon sequences. The sequenceof such oligonucleotide primers are, therefore, preferably derived fromtub intron sequence so that the entire exon (or coding region) can beanalyzed, as discussed below. Primer pairs useful for amplification oftub exons are preferably derived from adjacent introns. For example, inorder to amplify tub exon 5, a forward primer derived from the tubintron upstream of exon 5 (i.e., the intron between tub exon 4 and 5)could be used in conjunction with a reverse primer derived from the tubintron downstream of exon 5 (i.e., the intron between tub exon 5 and 6).

[0104] Appropriate primer pairs can be chosen such that each of thetwelve tub exons are amplified. FIG. 10A-10G depicts each of human tubexons 4 through 12 and, further, depicts intron sequences flanking eachof these exons. Primers for the amplification of tub exons can routinelybe designed by one of skill in the art by utilizing such intron flankingsequence.

[0105] As an example, and not by way of limitation, Table I, below,lists primers and primer pairs which can be utilized for theamplification of each of human tub exons 2 through 12. In this table, aprimer pair is listed for each of exons 2 through 12, which consists ofa forward primer derived from intron sequence upstream of the exon to beamplified and a reverse primer derived from sequence downstream of theexon to be amplified. Each of the primer pairs can be utilized,therefore, as part of a standard PCR reaction to amplify an individualtub exon. For each of the primer pairs listed in Table I, theapproximate size of the resulting amplified exon-containing fragment islisted. Utilizing the primer pairs of Table I to amplify human tub exon5, for example, primers F5 (the forward primer) and R5 (the reverseprimer) would be used to amplify a fragment of approximately 250 basepairs that would contain the entire coding region of exon 5. TABLE IAMPLIFIED HUMAN TUB PRIMER NAME FRAGMENT EXON AND SEQUENCE SIZE 2 F25′-GTT CAA GCT GGT F2/R2 = 200 bp TTC AAG ATG-3′ R2 5′-ATC ATC CAG GGAAGA TGG AC-3′ 3 F3 5′-CTT CCT GGT GGA F3/R3 = 220 bp GGC AGT G-3′ R35′-GAA GCA GTG ACG GGA TGT GG-3′ 4 F4 5′-GGG TAC CGA GCT F4/R4 = 295 bpCTG GTC-3′ R4 5′-TCC AAG TCA GGA GGA CAA AC-3′ 5 F5 5′-GAA AGT GCA TCTF5/R5 = 250 bp GAG AAC CTG-3′ R5 5′-CCT CCT CCT GGA TGT AAC TC-3′ 6 F65′-TGT GAC CAT GTG F6/R6 = 234 bp TAT TTC AGG-3′ R6 5″-CCT CTA ACG GATGAG CAG TC-3′ 7 F7 5′-GAT TTG GAT CCC F7/R7 = 331 bp AGA CCA CC-3′ R75′-GAC TTC CAG TCA CAT TTC AGC-3′ 8 F8 5′-GTG CAG ACC AGA F8/R8 = 300 bpGGC TGA G-3′ R8 5′-TTC AGG CCC TCT ACA GAC AG-3′ 9 F9 5′-TCA TAG GAC AGAF9/R9 = 210 bp CGA TGA GC-3′ R9 5′-GTC CTG GAT TTC ATA TCT ACC-3′ 10 F105′-AGG TAA ATA GAC F10/R10 = 218 bp GCC TCA GG-3′ R10 5′-ACG TCT GCC CTTAGA AGC TC-3′ 11 F11 5′-CTG GAC CTG GCT F11/R11 = 400 bp CAG GTG-3′ R115′-GTC ATT AGG GTT AGA AAG TTC C-3′ 12 F12 5′-TCT TCC CTC ATG F12/R12 =300 bp TGG TTT GG-3′ R12 5′-CCA CAG GCA GGC AGG CAA G-3′

[0106] Additional tub nucleic acid sequences which are preferred forsuch amplification-related analyses are those which will detect thepresence of the tub gene splice site mutation described, below, inSection 10.2 and depicted in FIG. 7A-7D.

[0107] Further, well-known genotyping techniques can be performed totype polymorphisms that are in close proximity to mutations in the tubgene itself. These polymorphisms can be used to identify individuals infamilies likely to carry mutations. If a polymorphism exhibits linkagedisequilibrium with mutations in the tub gene, it can also be used toidentify individuals in the general population likely to carrymutations. Polymorphisms that can be used in this way includerestriction fragment length polymorphisms (RFLPs), which involvesequence variations in restriction enzyme target sequences, single-basepolymorphisms and simple sequence repeat polymorphisms (SSLPs).

[0108] For example, Weber (U.S. Pat. No. 5,075,217, which isincorporated herein by reference in its entirety) describes a DNA markerbased on length polymorphisms in blocks of (dC-dA)n-(dG-dT)n shorttandem repeats. The average separation of (dC-dA)n-(dG-dT)n blocks isestimated to be 30,000-60,000 bp. Markers which are so closely spacedexhibit a high frequency co-inheritance, and are extremely useful in theidentification of genetic mutations, such as, for example, mutationswithin the tub gene, and the diagnosis of diseases and disorders relatedto tub mutations.

[0109] Also, Caskey et al. (U.S. Pat. No. 5,364,759, which isincorporated herein by reference in its entirety) describe a DNAprofiling assay for detecting short tri and tetra nucleotide repeatsequences. The process includes extracting the DNA of interest, such asthe tub gene, amplifying the extracted DNA, and labelling the repeatsequences to form a genotypic map of the individual's DNA.

[0110] A tub probe could additionally be used to directly identifyRFLPs. Additionally, a tub probe or primers derived from the tubsequence could be used to isolate genomic clones such as YACS, BACS,PACs, cosmids, phage or plasmids. The DNA contained in these clones canbe screened for single-base polymorphisms or simple sequence lengthpolymorphisms (SSLPs) using standard hybridization or sequencingprocedures.

[0111] Alternative diagnostic methods for the detection of tubgene-specific mutations or polymorphisms can include hybridizationtechniques which involve for example, contacting and incubating nucleicacids including recombinant DNA molecules, cloned genes or degeneratevariants thereof, obtained from a sample, e.g., derived from a patientsample or other appropriate cellular source, with one or more labelednucleic acid reagents including recombinant DNA molecules, cloned genesor degenerate variants thereof, as described in Section 5.1, underconditions favorable for the specific annealing of these reagents totheir complementary sequences within the tub gene. Preferably, thelengths of these nucleic acid reagents are at least 15 to 30nucleotides. After incubation, all non-annealed nucleic acids areremoved from the nucleic acid:tub molecule hybrid. The presence ofnucleic acids which have hybridized, if any such molecules exist, isthen detected. Using such a detection scheme, the nucleic acid from thecell type or tissue of interest can be immobilized, for example, to asolid support such as a membrane, or a plastic surface such as that on amicrotiter plate or polystyrene beads. In this case, after incubation,non-annealed, labeled nucleic acid reagents of the type described inSection 5.1 are easily removed. Detection of the remaining, annealed,labeled tub nucleic acid reagents is accomplished using standardtechniques well-known to those in the art. The tub gene sequences towhich the nucleic acid reagents have annealed can be compared to theannealing pattern expected from a normal tub gene sequence in order todetermine whether a tub gene mutation is present.

[0112] Among the tub nucleic acid sequences which are preferred for suchhybridization analyses are those which will detect the presence of thetub gene splice site mutation described, below, in Section 10.2 anddepicted in FIG. 7A-7D.

[0113] Quantitative and qualitative aspects of tub gene expression canalso be assayed. For example, RNA from a cell type or tissue known, orsuspected, to express the tub gene, such as brain, especiallyhypothalamus cells, may be isolated and tested utilizing hybridizationor PCR techniques such as are described, above. The isolated cells canbe derived from cell culture or from a patient. The analysis of cellstaken from culture may be a necessary step in the assessment of cells tobe used as part of a cell-based gene therapy technique or,alternatively, to test the effect of compounds on the expression of thetub gene. Such analyses may reveal both quantitative and qualitativeaspects of the expression pattern of the tub gene, including activationor inactivation of tub gene expression and presence of alternativelyspliced tub transcripts.

[0114] In one embodiment of such a detection scheme, a cDNA molecule issynthesized from an RNA molecule of interest (e.g., by reversetranscription of the RNA molecule into cDNA). All or part of theresulting cDNA is then used as the template for a nucleic acidamplification reaction, such as a PCR amplification reaction, or thelike. The nucleic acid reagents used as synthesis initiation reagents(e.g., primers) in the reverse transcription and nucleic acidamplification steps of this method are chosen from among the tub genenucleic acid reagents described in Section 5.1. The preferred lengths ofsuch nucleic acid reagents are at least 9-30 nucleotides.

[0115] For detection of the amplified product, the nucleic acidamplification may be performed using radioactively or non-radioactivelylabeled nucleotides. Alternatively, enough amplified product may be madesuch that the product may be visualized by standard ethidium bromidestaining or by utilizing any other suitable nucleic acid stainingmethod.

[0116] Such RT-PCR techniques can be utilized to detect differences intub transcript size which may be due to normal or abnormal alternativesplicing. Additionally, such techniques can be performed using standardtechniques to detect quantitative differences between levels of fulllength and/or alternatively spliced tub transcripts detected in normalindividuals relative to those individuals exhibiting body weightdisorders or exhibiting a predisposition to toward such body weightdisorders.

[0117] In the case where detection of specific alternatively splicedspecies is desired, appropriate primers and/or hybridization probes canbe used. Using the detection of transcripts containing tub exon 5, forexample, appropriate amplification primers can be chosen which will onlyyield an amplified fragment using cDNA derived from an exon 5-containingtranscript. One of the primer pairs can be designed, for example, tospecifically utilize an exon 5 sequence. In the absence of suchsequence, no amplification would occur. Alternatively, primer pairs maybe chosen utilizing the sequence data depicted in FIGS. 6A-6D and 9A-9Dto choose primers which will yield fragments of differing size dependingon whether exon 5 is present or absent from the transcript tubtranscript being utilized.

[0118] As an alternative to amplification techniques, standard Northernanalyses can be performed if a sufficient quantity of the appropriatecells can be obtained. Utilizing such techniques, quantitative as wellas size related differences between tub transcripts can also bedetected.

[0119] Additionally, it is possible to perform such tub gene expressionassays “in situ”, i.e., directly upon tissue sections (fixed and/orfrozen) of patient tissue obtained from biopsies or resections, suchthat no nucleic acid purification is necessary. Nucleic acid reagentssuch as those described in Section 5.1 may be used as probes and/orprimers for such in situ procedures (see, for example, Nuovo, G. J.,1992, “PCR In Situ Hybridization: Protocols And Applications”, RavenPress, NY).

5.4.1.2. Detection of Tub Gene Products

[0120] Antibodies directed against wild type or mutant tub gene productsor conserved variants or peptide fragments thereof, which are discussed,above, in Section 5.3, may also be used as body weight disorderdiagnostics and prognostics, as described herein. Such diagnosticmethods, may be used to detect abnormalities in the level of tub geneexpression, or abnormalities in the structure and/or temporal, tissue,cellular, or subcellular location of tub gene product. Because evidencedisclosed herein indicates that the tub gene product is an intracellulargene product, the antibodies and immunoassay methods described belowhave important in vitro applications in assessing the efficacy oftreatments for body-weight disorders such as obesity, cachexia andanorexia. Antibodies, or fragments of antibodies, such as thosedescribed below, may be used to screen potentially therapeutic compoundsin vitro to determine their effects on tub gene expression and tubpeptide production. The compounds which have beneficial effects on bodyweight disorders, such as obesity, cachexia and anorexia, can beidentified, and a therapeutically effective dose determined.

[0121] In vitro immunoassays may also be used, for example, to assessthe efficacy of cell-based gene therapy for body weight disorders,including obesity, cachexia and anorexia. Antibodies directed againsttub peptides may be used in vitro to determine the level of tub geneexpression achieved in cells genetically engineered to produce tubpeptides. Given that evidence disclosed herein indicates that the tubgene product is an intracellular gene product, such an assessment is,preferably, done using cell lysates or extracts. Such analysis willallow for a determination of the number of transformed cells necessaryto achieve therapeutic efficacy in vivo, as well as optimization of thegene replacement protocol.

[0122] The tissue or cell type to be analyzed will generally includethose which are known, or suspected, to express the tub gene, such as,for example, hypothalamic cells. The protein isolation methods employedherein may, for example, be such as those described in Harlow and Lane(Harlow, E. and Lane, D., 1988, “Antibodies: A Laboratory Manual”, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which isincorporated herein by reference in its entirety. The isolated cells canbe derived from cell culture or from a patient. The analysis of celltaken from culture may be a necessary step in the assessment of cells tobe used as part of a cell-based gene therapy technique or,alternatively, to test the effect of compounds on the expression of thetub gene.

[0123] Preferred diagnostic methods for the detection of tub geneproducts or conserved variants or peptide fragments thereof, mayinvolve, for example, immunoassays wherein the tub gene products orconserved variants, including gene products which are the result ofalternatively spliced transcripts, or peptide fragments are detected bytheir interaction with an anti-tub gene product-specific antibody.

[0124] For example, antibodies, or fragments of antibodies, such asthose described, above, in Section 5.3, useful in the present inventionmay be used to quantitatively or qualitatively detect the presence oftub gene products or conserved variants or peptide fragments thereof.This can be accomplished, for example, by immunofluorescence techniquesemploying a fluorescently labeled antibody (see below, this Section)coupled with light microscopic, flow cytometric, or fluorimetricdetection. Such techniques are especially preferred if such tub geneproducts are expressed on the cell surface.

[0125] The antibodies (or fragments thereof) useful in the presentinvention may, additionally, be employed histologically, as inimmunofluorescence or immunoelectron microscopy, for in situ detectionof tub gene products or conserved variants or peptide fragments thereof.In situ detection may be accomplished by removing a histologicalspecimen from a patient, and applying thereto a labeled antibody of thepresent invention. The antibody (or fragment) is preferably applied byoverlaying the labeled antibody (or fragment) onto a biological sample.Through the use of such a procedure, it is possible to determine notonly the presence of the tub gene product, or conserved variants orpeptide fragments, but also its distribution in the examined tissue.Using the present invention, those of ordinary skill will readilyperceive that any of a wide variety of histological methods (such asstaining procedures) can be modified in order to achieve such in situdetection.

[0126] Immunoassays for tub gene products or conserved variants orpeptide fragments thereof will typically comprise incubating a sample,such as a biological fluid, a tissue extract, freshly harvested cells,or lysates of cells which have been incubated in cell culture, in thepresence of a detectably labeled antibody capable of identifying tubgene products or conserved variants or peptide fragments thereof, anddetecting the bound antibody by any of a number of techniques well-knownin the art.

[0127] The biological sample may be brought in contact with andimmobilized onto a solid phase support or carrier such asnitrocellulose, or other solid support which is capable of immobilizingcells, cell particles or soluble proteins. The support may then bewashed with suitable buffers followed by treatment with the detectablylabeled tub gene specific antibody. The solid phase support may then bewashed with the buffer a second time to remove unbound antibody. Theamount of bound label on solid support may then be detected byconventional means.

[0128] By “solid phase support or carrier” is intended any supportcapable of binding an antigen or an antibody. Well-known supports orcarriers include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support configuration maybe spherical, as in a bead, or cylindrical, as in the inside surface ofa test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, etc. Preferred supportsinclude polystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

[0129] The binding activity of a given lot of anti-tub gene productantibody may be determined according to well known methods. Thoseskilled in the art will be able to determine operative and optimal assayconditions for each determination by employing routine experimentation.

[0130] One of the ways in which the tub gene peptide-specific antibodycan be detectably labeled is by linking the same to an enzyme and use inan enzyme immunoassay (EIA) (Voller, A., “The Enzyme LinkedImmunosorbent Assay (ELISA)”, 1978, Diagnostic Horizons 2:1-7,Microbiological Associates Quarterly Publication, Walkersville, Md.);Voller, A. et al., 1978, J. Clin. Pathol. 31:507-520; Butler, J. E.,1981, Meth. Enzymol. 73:482-523; Maggio, E. (ed.), 1980, EnzymeImmunoassay, CRC Press, Boca Raton, Fla.,; Ishikawa, E. et al., (eds.),1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The enzyme which is boundto the antibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietywhich can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes which can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

[0131] Detection may also be accomplished using any of a variety ofother immunoassays. For example, by radioactively labeling theantibodies or antibody fragments, it is possible to detect tub genepeptides through the use of a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986,which is incorporated by reference herein). The radioactive isotope canbe detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography.

[0132] It is also possible to label the antibody with a fluorescentcompound. When the fluorescently labeled antibody is exposed to light ofthe proper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

[0133] The antibody can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²Eu, or others of the lanthanide series. Thesemetals can be attached to the antibody using such metal chelating groupsas diethylenetriaminepentacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

[0134] The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

[0135] Likewise, a bioluminescent compound may be used to label theantibody of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in, which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

5.4.2. Screening Assays for Compounds that Modulate Tub Gene Activity

[0136] The following assays are designed to identify compounds that bindto tub gene products, bind to other intracellular proteins that interactwith a tub gene product, to compounds that interfere with theinteraction of the tub gene product with other intracellular proteinsand to compounds which modulate the activity of tub gene (i.e., modulatethe level of tub gene expression and/or modulate the level of tub geneproduct activity). Assays may additionally be utilized which identifycompounds which bind to tub gene regulatory sequences (e.g., promotersequences). See e.g., Platt, K. A., 1994, J. Biol. Chem.269:28558-28562, which is incorporated herein by reference in itsentirety, which may modulate the level of tub gene expression. Compoundsmay include, but are not limited to, small organic molecules which areable to cross the blood-brain barrier, gain entry into an appropriatecell and affect expression of the tub gene or some other gene involvedin the body weight regulatory pathway, or other intracellular proteins.Methods for the identification of such intracellular proteins aredescribed, below, in Section 5.4.2.2. Such intracellular proteins may beinvolved in the control and/or regulation of body weight. Further, amongthese compounds are compounds which affect the level of tub geneexpression and/or tub gene product activity and which can be used in thetherapeutic treatment of body weight disorders, including obesity,cachexia and anorexia, as described, below, in Section 5.4.3.

[0137] Compounds may include, but are not limited to, peptides such as,for example, soluble peptides, including but not limited to, Ig-tailedfusion peptides, and members of random peptide libraries; (see, e.g.,Lam, K. S. et al., 1991, Nature 354:82-84; Houghten, R. et al., 1991,Nature 354:84-86), and combinatorial chemistry-derived molecular librarymade of D- and/or L-configuration amino acids, phosphopeptides(including, but not limited to members of random or partiallydegenerate, directed phosphopeptide libraries; see, e.g., Songyang, Z.et al., 1993, Cell 72:767-778), antibodies (including, but not limitedto, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric orsingle chain antibodies, and FAb, F(ab′)₂ and FAb expression libraryfragments, and epitope-binding fragments thereof), and small organic orinorganic molecules.

[0138] Compounds identified via assays such as those described hereinmay be useful, for example, in elaborating the biological function ofthe tub gene product, and for ameliorating body weight disorders. Assaysfor testing the effectiveness of compounds, identified by, for example,techniques such as those described in Section 5.4.2.1-5.4.2.3, arediscussed, below, in Section 5.4.2.4.

5.4.2.1. In Vitro Screening Assays for Compounds that Bind to the TubGene Product

[0139] In vitro systems may be designed to identify compounds capable ofbinding the tub gene products of the invention. Compounds identified maybe useful, for example, in modulating the activity of wild type and/ormutant tub gene products, may be useful in elaborating the biologicalfunction of the tub gene product, may be utilized in screens foridentifying compounds that disrupt normal tub gene product interactions,or may in themselves disrupt such interactions.

[0140] The principle of the assays used to identify compounds that bindto the tub gene product involves preparing a reaction mixture of the tubgene product and the test compound under conditions and for a timesufficient to allow the two components to interact and bind, thusforming a complex which can be removed and/or detected in the reactionmixture. These assays can be conducted in a variety of ways. Forexample, one method to conduct such an assay would involve anchoring tubgene product or the test substance onto a solid phase and detecting tubgene product/test compound complexes anchored on the solid phase at theend of the reaction. In one embodiment of such a method, the tub geneproduct may be anchored onto a solid surface, and the test compound,which is not anchored, may be labeled, either directly or indirectly.

[0141] In practice, microtiter plates may conveniently be utilized asthe solid phase. The anchored component may be immobilized bynon-covalent or covalent attachments. Non-covalent attachment may beaccomplished by simply coating the solid surface with a solution of theprotein and drying. Alternatively, an immobilized antibody, preferably amonoclonal antibody, specific for the protein to be immobilized may beused to anchor the protein to the solid surface. The surfaces may beprepared in advance and stored.

[0142] In order to conduct the assay, the nonimmobilized component isadded to the coated surface containing the anchored component. After thereaction is complete, unreacted components are removed (e.g., bywashing) under conditions such that any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thepreviously nonimmobilized component is pre-labeled, the detection oflabel immobilized on the surface indicates that complexes were formed.Where the previously nonimmobilized component is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface;e.g., using a labeled antibody specific for the previouslynonimmobilized component (the antibody, in turn, may be directly labeledor indirectly labeled with a labeled anti-Ig antibody).

[0143] Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for tub geneproduct or the test compound to anchor any complexes formed in solution,and a labeled antibody specific for the other component of the possiblecomplex to detect anchored complexes.

5.4.2.2. Assays for Intracellular Proteins that Interact with the TubGene Product

[0144] Any method suitable for detecting protein-protein interactionsmay be employed for identifying tub protein-intracellular proteininteractions.

[0145] Among the traditional methods which may be employed areco-immunoprecipitation, crosslinking and co-purification throughgradients or chromatographic columns. Utilizing procedures such as theseallows for the identification of intracellular proteins which interactwith tub gene products. Once isolated, such an intracellular protein canbe identified and can, in turn, be used, in conjunction with standardtechniques, to identify proteins it interacts with. For example, atleast a portion of the amino acid sequence of the intracellular proteinwhich interacts with the tub gene product can be ascertained usingtechniques well known to those of skill in the art, such as via theEdman degradation technique (see, e.g., Creighton, 1983, “Proteins:Structures and Molecular Principles”, W. H. Freeman & Co., N.Y.,pp.34-49). The amino acid sequence obtained may be used as a guide forthe generation of oligonucleotide mixtures that can be used to screenfor gene sequences encoding such intracellular proteins. Screening madebe accomplished, for example, by standard hybridization or PCRtechniques. Techniques for the generation of oligonucleotide mixturesand the screening are well-known. (See, e.g., Ausubel, supra., and PCRProtocols: A Guide to Methods and Applications, 1990, Innis, M. et al.,eds. Academic Press, Inc., New York).

[0146] Additionally, methods may be employed which result in thesimultaneous identification of genes which encode the intracellularprotein interacting with the tub protein. These methods include, forexample, probing expression libraries with labeled tub protein, usingtub protein in a manner similar to the well known technique of antibodyprobing of λgt11 libraries.

[0147] One method which detects protein interactions in vivo, thetwo-hybrid system, is described in detail for illustration only and notby way of limitation. One version of this system has been described(Chien et al., 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and iscommercially available from Clontech (Palo Alto, Calif.).

[0148] Briefly, utilizing such a system, plasmids are constructed thatencode two hybrid proteins: one consists of the DNA-binding domain of atranscription activator protein fused to the tub gene product and theother consists of the transcription activator protein's activationdomain fused to an unknown protein that is encoded by a cDNA which hasbeen recombined into this plasmid as part of a cDNA library. TheDNA-binding domain fusion plasmid and the cDNA library are transformedinto a strain of the yeast Saccharomyces cerevisiae that contains areporter gene (e.g., HBS or lacZ) whose regulatory region contains thetranscription activator's binding site. Either hybrid protein alonecannot activate transcription of the reporter gene: the DNA-bindingdomain hybrid cannot because it does not provide activation function andthe activation domain hybrid cannot because it cannot localize to theactivator's binding sites. Interaction of the two hybrid proteinsreconstitutes the functional activator protein and results in expressionof the reporter gene, which is detected by an assay for the reportergene product.

[0149] The two-hybrid system or related methodology may be used toscreen activation domain libraries for proteins that interact with the“bait” gene product. By way of example, and not by way of limitation,tub gene products may be used as the bait gene product. Total genomic orcDNA sequences are fused to the DNA encoding an activation domain. Thislibrary and a plasmid encoding a hybrid of a bait tub gene product fusedto the DNA-binding domain are cotransformed into a yeast reporterstrain, and the resulting transformants are screened for those thatexpress the reporter gene. For example, and not by way of limitation, abait tub gene sequence, such as the 1.5 kb open reading frame of the tubgene, as depicted in FIG. 6A-6D or FIG. 9A-9D can be cloned into avector such that it is translationally fused to the DNA encoding theDNA-binding domain of the GAL4 protein. These colonies are purified andthe library plasmids responsible for reporter gene expression areisolated. DNA sequencing is then used to identify the proteins encodedby the library plasmids.

[0150] A cDNA library of the cell line from which proteins that interactwith bait tub gene product are to be detected can be made using methodsroutinely practiced in the art. According to the particular systemdescribed herein, for example, the cDNA fragments can be inserted into avector such that they - are translationally fused to the transcriptionalactivation domain of GAL4. This library can be co-transformed along withthe bait tub gene-GAL4 fusion plasmid into a yeast strain which containsa lacZ gene driven by a promoter which contains GAL4 activationsequence. A cDNA encoded protein, fused to GAL4 transcriptionalactivation domain, that interacts with bait tub gene product willreconstitute an active GAL4 protein and thereby drive expression of theHIS3 gene. Colonies which express HIS3 can be detected by their growthon petri dishes containing semi-solid agar based media lackinghistidine. The cDNA can then be purified from these strains, and used toproduce and isolate the bait tub gene-interacting protein usingtechniques routinely practiced in the art.

5.4.2.3. Assays for Compounds that Interfere with Tub GeneProduct/Intracellular Macromolecule Interaction

[0151] The tub gene products of the invention may, in vivo, interactwith one or more intracellular macromolecules, such as proteins. Suchmacromolecules may include, but are not limited to, nucleic acidmolecules and those proteins identified via methods such as thosedescribed, above, in Section 5.4.2.2. For purposes of this discussion,such intracellular macromolecules are referred to herein as “bindingpartners”. Compounds that disrupt tub binding in this way may be usefulin regulating the activity of the tub gene product, especially mutanttub gene products. Such compounds may include, but are not limited tomolecules such as peptides, and the like, as described, for example, inSection 5.4.2.1. above, which would be capable of gaining access to theintracellular tub gene product.

[0152] The basic principle of the assay systems used to identifycompounds that interfere with the interaction between the tub geneproduct and its intracellular binding partner or partners involvespreparing a reaction mixture containing the tub gene product, and thebinding partner under conditions and for a time sufficient to allow thetwo to interact and bind, thus forming a complex. In order to test acompound for inhibitory activity, the reaction mixture is prepared inthe presence and absence of the test compound. The test compound may beinitially included in the reaction mixture, or may be added at a timesubsequent to the addition of tub gene product and its intracellularbinding partner. Control reaction mixtures are incubated without thetest compound or with a placebo. The formation of any complexes betweenthe tub gene protein and the intracellular binding partner is thendetected. The formation of a complex in the control reaction, but not inthe reaction mixture containing the test compound, indicates that thecompound interferes with the interaction of the tub gene protein and theinteractive binding partner. Additionally, complex formation withinreaction mixtures containing the test compound and normal tub geneprotein may also be compared to complex formation within reactionmixtures containing the test compound and a mutant tub gene protein.This comparison may be important in those cases wherein it is desirableto identify compounds that disrupt interactions of mutant but not normaltub gene proteins.

[0153] The assay for compounds that interfere with the interaction ofthe tub gene products and binding partners can be conducted in aheterogeneous or homogeneous format. Heterogeneous assays involveanchoring either the tub gene product or the binding partner onto asolid phase and detecting complexes anchored on the solid phase at theend of the reaction. In homogeneous assays, the entire reaction iscarried out in a liquid phase. In either approach, the order of additionof reactants can be varied to obtain different information about thecompounds being tested. For example, test compounds that interfere withthe interaction between the tub gene products and the binding partners,e.g., by competition, can be identified by conducting the reaction inthe presence of the test substance; i.e., by adding the test substanceto the reaction mixture prior to or simultaneously with the tub geneprotein and interactive intracellular binding partner. Alternatively,test compounds that disrupt preformed complexes, e.g. compounds withhigher binding constants that displace one of the components from thecomplex, can be tested by adding the test compound to the reactionmixture after complexes have been formed. The various formats aredescribed briefly below.

[0154] In a heterogeneous assay system, either the tub gene product orthe interactive intracellular binding partner, is anchored onto a solidsurface, while the non-anchored species is labeled, either directly orindirectly. In practice, microtiter plates are conveniently utilized.The anchored species may be immobilized by non-covalent or covalentattachments. Non-covalent attachment may be accomplished simply bycoating the solid surface with a solution of the tub gene product orbinding partner and drying. Alternatively, an immobilized antibodyspecific for the species to be anchored may be used to anchor thespecies to the solid surface. The surfaces may be prepared in advanceand stored.

[0155] In order to conduct the assay, the partner of the immobilizedspecies is exposed to the coated surface with or without the testcompound. After the reaction is complete, unreacted components areremoved (e.g., by washing) and any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thenon-immobilized species is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe non-immobilized species is not prelabeled, an indirect label can beused to detect complexes anchored on the surface; e.g., using a labeledantibody specific for the initially non-immobilized species (theantibody, in turn, may be directly labeled or indirectly labeled with alabeled anti-Ig antibody). Depending upon the order of addition ofreaction components, test compounds which inhibit complex formation orwhich disrupt preformed complexes can be detected.

[0156] Alternatively, the reaction can be conducted in a liquid phase inthe presence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

[0157] In an alternate embodiment of the invention, a homogeneous assaycan be used. In this approach, a preformed complex of the tub geneprotein and the interactive intracellular binding partner is prepared inwhich either the tub gene product or its binding partners is labeled,but the signal generated by the label is quenched due to complexformation (see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein whichutilizes this approach for immunoassays). The addition of a testsubstance that competes with and displaces one of the species from thepreformed complex will result in the generation of a signal abovebackground. In this way, test substances which disrupt tub geneprotein/intracellular binding partner interaction can be identified.

[0158] In a particular embodiment, the tub gene product can be preparedfor immobilization using recombinant DNA techniques described in Section5.2. above. For example, the tub coding region can be fused to aglutathione-S-transferase (GST) gene using a fusion vector, such aspGEX-5X-1, in such a manner that its binding activity is maintained inthe resulting fusion protein. The interactive intracellular bindingpartner can be purified and used to raise a monoclonal antibody, usingmethods routinely practiced in the art and described above, in Section5.3. This antibody can be labeled with the radioactive isotope ¹²⁵I, forexample, by methods routinely practiced in the art. In a heterogeneousassay, e.g., the GST-tub fusion protein can be anchored toglutathione-agarose beads. The interactive intracellular binding partnercan then be added in the presence or absence of the test compound in amanner that allows interaction and binding to occur. At the end of thereaction period, unbound material can be washed away, and the labeledmonoclonal antibody can be added to the system and allowed to bind tothe complexed components. The interaction between the tub gene proteinand the interactive intracellular binding partner can be detected bymeasuring the amount of radioactivity that remains associated with theglutathione-agarose beads. A successful inhibition of the interaction bythe test compound will result in a decrease in measured radioactivity.

[0159] Alternatively, the GST-tub gene fusion protein and theinteractive intracellular binding partner can be mixed together inliquid in the absence of the solid glutathione-agarose beads. The testcompound can be added either during or after the species are allowed tointeract. This mixture can then be added to the glutathione-agarosebeads and unbound material is washed away. Again the extent ofinhibition of the tub gene product/binding partner interaction can bedetected by adding the labeled antibody and measuring the radioactivityassociated with the beads.

[0160] In another embodiment of the invention, these same techniques canbe employed using peptide fragments that correspond to the bindingdomains of the tub protein and/or the interactive intracellular orbinding partner (in cases where the binding partner is a protein), inplace of one or both of the full length proteins. Any number of methodsroutinely practiced in the art can be used to identify and isolate thebinding sites. These methods include, but are not limited to,mutagenesis of the gene encoding one of the proteins and screening fordisruption of binding in a co-immunoprecipitation assay. Compensatingmutations in the gene encoding the second species in the complex canthen be selected. Sequence analysis of the genes encoding the respectiveproteins will reveal the mutations that correspond to the region of theprotein involved in interactive binding. Alternatively, one protein canbe anchored to a solid surface using methods described in this Sectionabove, and allowed to interact with and bind to its labeled bindingpartner, which has been treated with a proteolytic enzyme, such astrypsin. After washing, a short, labeled peptide comprising the bindingdomain may remain associated with the solid material, which can beisolated and identified by amino acid sequencing. Also, once the genecoding for the intracellular binding partner is obtained, short genesegments can be engineered to express peptide fragments of the protein,which can then be tested for binding activity and purified orsynthesized.

[0161] For example, and not by way of limitation, a tub gene product canbe anchored to a solid material as described, above, in this Section bymaking a GST-tub fusion protein and allowing it to bind to glutathioneagarose beads. The interactive intracellular binding partner can belabeled with a radioactive isotope, such as ³⁵S, and cleaved with aproteolytic enzyme such as trypsin. Cleavage products can then be addedto the anchored GST-tub fusion protein and allowed to bind. Afterwashing away unbound peptides, labeled bound material, representing theintracellular binding partner binding domain, can be eluted, purified,and analyzed for amino acid sequence by well-known methods. Peptides soidentified can be produced synthetically or fused to appropriatefacilitative proteins using recombinant DNA technology.

5.4.2.4. Assays for Identification of Compounds that Ameliorate BodyWeight Disorders

[0162] Compounds, including but not limited to binding compoundsidentified via assay techniques such as those described, above, inSections 5.4.2.1-5.4.2.3, can be tested for the ability to amelioratebody weight disorder symptoms, including obesity. It should be notedthat although tub gene products are intracellular molecules which arenot secreted and have no transmembrane component, the assays describedherein can identify compounds which affect tub gene activity by eitheraffecting tub gene expression or by affecting the level of tub geneproduct activity. For example, compounds may be identified which areinvolved in another step in the pathway in which the tub gene and/or tubgene product is involved and, by affecting this same pathway maymodulate the affect of tub on the development of body weight disorders.Such compounds can be used as part of a therapeutic method for thetreatment of body weight disorders.

[0163] Described below are cell-based and animal model-based assays forthe identification of compounds exhibiting such an ability to amelioratebody weight disorder symptoms.

[0164] First, cell-based systems can be used to identify compounds whichmay act to ameliorate body weight disorder symptoms. Such cell systemscan include, for example, recombinant or non-recombinant cell, such ascell lines, which express the tub gene. For example, hypothalamus cells,such as, for example GH-1 (Melcang; R. C. et al., 1995, Endocrinology136:679-686) and GN (Radovick, S. et al., 1991, Proc. Natl. Acad. Sci.USA 88:3402-3406) hypothalamic cell lines can be used.

[0165] In utilizing such cell systems, cells may be exposed to acompound, suspected of exhibiting an ability to ameliorate body weightdisorder symptoms, at a sufficient concentration and for a timesufficient to elicit such an amelioration of body weight disordersymptoms in the exposed cells. After exposure, the cells can be assayedto measure alterations in the expression of the tub gene, e.g., byassaying cell lysates for tub mRNA transcripts (e.g., by Northernanalysis) or for tub protein expressed in the cell; compounds whichincrease expression of the tub gene are good candidates as therapeutics.Alternatively, the cells are examined to determine whether one or morebody weight disorder-like cellular phenotypes has been altered toresemble a more normal or more wild type, non-body weight disorderphenotype, or a phenotype more likely to produce a lower incidence orseverity of disorder symptoms.

[0166] In addition, animal-based body weight disorder systems, which mayinclude, for example tub mice, may be used to identify compounds capableof ameliorating body weight disorder-like symptoms. Such animal modelsmay be used as test substrates for the identification of drugs,pharmaceuticals, therapies and interventions which may be effective intreating such disorders. For example, animal models may be exposed to acompound, suspected of exhibiting an ability to ameliorate body weightdisorder symptoms, at a sufficient concentration and for a timesufficient to elicit such an amelioration of body weight disordersymptoms in the exposed animals. The response of the animals to theexposure may be monitored by assessing the reversal of disordersassociated with body weight disorders such as obesity.

[0167] With regard to intervention, any treatments which reverse anyaspect of body weight disorder-like symptoms should be considered ascandidates for human body weight disorder therapeutic intervention.Dosages of test agents may be determined by deriving dose-responsecurves, as discussed in Section 5.5.1, below.

5.4.3. Compounds and Methods for the Treatment of Body Weight

[0168] Described below are methods and compositions whereby body weightincluding body weight disorders, including obesity, cachexia andanorexia may be treated. Because a loss of normal tub gene productfunction results in the development of an obese phenotype, an increasein tub gene product activity would facilitate progress towards a normalbody weight state in individuals exhibiting a deficient level of tubgene expression and/or tub gene product activity.

[0169] Alternatively, symptoms of certain body weight disorders such as,for example, cachexia, which involve a lower than normal body weightphenotype, may be ameliorated by decreasing the level of tub geneexpression and/or tub gene product activity. For example, tub genesequences may be utilized in conjunction with well-known antisense, gene“knock-out,” ribozyme and/or triple helix methods to decrease the levelof tub gene expression. Such methods can also be useful for agriculturalapplications in which a more favorable fat:level body mass ratio (i.e.,a decreased ratio) is desired.

[0170] With respect to an increase in the level of normal tub geneexpression and/or tub gene product activity, tub gene nucleic acidsequences, described, above, in Section 5.1, can, for example, beutilized for the treatment of body weight disorders, including obesity.Such treatment can be administered, for example, in the form of genereplacement therapy. Specifically, one or more copies of a normal tubgene or a portion of the tub gene that directs the production of a tubgene product exhibiting normal tub gene function, may be inserted intothe appropriate cells within a patient, using vectors which include, butare not limited to adenovirus, adeno-associated virus, and retrovirusvectors, in addition to other particles that introduce DNA into cells,such as liposomes.

[0171] Because the tub gene is expressed in the brain, including thehypothalamus, such gene replacement therapy techniques should be capabledelivering tub gene sequences to these cell types within patients. Thus,the techniques for delivery of tub gene sequences should be able toreadily cross the blood-brain barrier, which are well known to those ofskill in the art (see, e.g., PCT application, publication No.WO89/10134, which is incorporated herein by reference in its entirety),or, alternatively, should involve direct administration of such tub genesequences to the site of the cells in which the tub gene sequences areto be expressed. With respect to delivery which is capable of crossingthe blood-brain barrier, viral vectors such as, for example, thosedescribed above, are preferable.

[0172] Additional methods which may be utilized to increase the overalllevel of tub gene expression and/or tub gene product activity includethe introduction of appropriate tub-expressing cells, preferablyautologous cells, into a patient at positions and in numbers which aresufficient to ameliorate the symptoms of body weight disorders,including obesity. Such cells may be either recombinant ornon-recombinant.

[0173] Among the cells which can be administered to increase the overalllevel of tub gene expression in a patient are normal cells, preferablyhypothalamus cells, which express the tub gene. Among the hypothalamiccells which can be administered are hypothalamic cell lines, whichinclude, but are not limited to the GT1-1 cell line (Melcangi, R. C. etal., 1995, Endocrin. 136:679-686).

[0174] Alternatively, cells, preferably autologous cells, can beengineered to express tub gene sequences which may then be introducedinto a patient in positions appropriate for the amelioration of bodyweight disorder symptoms. Alternately, cells which express the tub genein a wild type in MHC matched individuals, i.e., non-tub individual, andmay include, for example, hypothalamic cells. The expression of the tubgene sequences is controlled by the appropriate gene regulatorysequences to allow such expression in the necessary cell types. Suchgene regulatory sequences are well known to the skilled artisan. Suchcell-based gene therapy techniques are well known to those skilled inthe art, see, e.g., Anderson, F., U.S. Pat. No. 5,399,349.

[0175] When the cells to be administered are non-autologous cells, theycan be administered using well known techniques which prevent a hostimmune response against the introduced cells from developing. Forexample, the cells may be introduced in an encapsulated form which,while allowing for an exchange of components with the immediateextracellular environment, does not allow the introduced cells to berecognized by the host immune system.

[0176] Additionally, compounds, such as those identified via techniquessuch as those described, above, in Section 5.4.2, which are capable ofmodulating tub gene product activity can be administered using standardtechniques which are well known to those of skill in the art. Ininstances in which the compounds to be administered are to involve aninteraction with brain cell types such as, for example, hypothalamiccell types, the administration techniques should include well known oneswhich allow for a crossing of the blood-brain barrier.

5.5. High-Throughput Screening Assays for Drugs Useful in Regulation ofBody Weight

[0177] At least two different assay systems, described in thesubsections below, can be designed and used as high-throughput screeningassays to identify compounds or compositions that modulate or alter tubgene product activity, and therefore, modulate weight control. Thescreening assays described herein may be used singly or in combinationwith other assays, including animal models, to identify compounds whichmodulate tub gene product activity.

[0178] The systems described below may be formulated into kits. To thisend, the tub gene product, either wild type or mutant, or cellsexpressing the tub gene product, either wild type or mutant, can bepackaged in a variety of containers, e.g., vials, tubes, microtitre wellplates, bottles and the like. Other reagents can be included in separatecontainers and provided with the kit, e.g., positive controls samples,negative controls samples, SH2 containing peptides and/or proteins,reporter constructs, buffers, cell culture media, etc.

[0179] In addition, animal-based systems or models for a body weightdisorder may be used to identify compounds capable of amelioratingsymptoms of the disorder. Such animal models may be used as testsubstrates for the identification of drug pharmaceuticals, therapies andinterventions, including compounds, small molecules, ribozymes andantisense molecules that may be effective in treating such disorders.Any compound tested in the high-throughput screening assays as may betested in animals. In particular, any compound identified in thehigh-throughput assays as altering tub gene product activity may furtherbe tested in an animal. For example animal models may be exposed to acompound suspected of exhibiting an ability to ameliorate symptoms, at asufficient concentration and for a sufficient time to elicit such anamelioration of a tub gene related disorder in the exposed animals. Theresponse of the animals to the exposure may be monitored by assessingthe reversal of such symptoms or any other way found suitable to assaythe effects of such compounds in animals or humans.

5.5.1. Cell-Based Assays

[0180] In accordance with the invention, a cell-based assay system canbe used to screen for compounds that modulate the activity of tub geneproduct and thereby, modulate body weight. To this end, cells, orlysates thereof, that endogenously express tub gene product, either wildtype or mutant, can be used to screen for compounds useful in thealteration or modulation of tub gene product activity. Cells isolatedfrom transgenic animals engineered to express the tub gene product orprimary cells expressing the tub gene product isolated from animal orhuman tissue may be used for screening purposes. Alternatively, celllines genetically engineered to express the tub gene product asdescribed in Section 5.2 above, or lysates thereof, may be used forscreening purposes. Preferably, host cells genetically engineered toexpress a functional insulin receptor and/or reporter genes regulated byinsulin or lysates thereof, may be used for screening purposes.

[0181] In utilizing such cell systems, the cells expressing tub geneproduct and an insulin receptor are exposed to a test compound or tovehicle controls (e.g., placebos). After exposure, no cells arestimulated with insulin or IGF-1 the cells can then be assayed tomeasure the expression and/or activity of components of the signaltransduction pathway of the insulin receptor, or the activity of thesignal transduction pathway itself can be assayed. In this regard, anyintermediate step in the signal transduction pathway can be measured orassayed to determine the effect of the test compound on the activity ofthe tub gene product in the signal transduction pathway. For example,after exposure, cell lysates can be assayed for induction ofphosphotidylinositol turnover. The ability of a test compound toincrease levels of phosphotidylinositol turnover measured by calciumflux, different than those levels seen with cells treated with a vehiclecontrol indicates that the test compound modulates signal transductionmediated by stimulation of tub gene product.

[0182] For example, to determine phosphotidylinositol turnover measuredby calcium flux, a bioluminescence assay may be utilized such as thosedescribed in Brownstein, I. et al. (1994, Biotechniques 17: 172-177).The assay utilizes cells, or lysates thereof, which have beentransfected with DNA vectors encoding the insulin receptor with orwithout DNA vectors encoding tub gene product. The cells are labeledwith a calcium sensitive bioluminescent protein. Test compounds orvehicle controls are added to the cells. The cells are stimulated withinsulin or insulin growth factor-1 (IGF-1) for approximately 30 minutes.The cells are assayed for bioluminescence. The assay may be performed ina 96 well-based plate to enable high-throughput screening. Such assaysprovide a simple, sensitive, easily automatable detection system forpharmaceutical screening.

[0183] In another embodiment, constructs containing an insulin orIGF-responsive element, such as the NPY promoter, linked to any of avariety of different reporter genes may be introduced into cellsexpressing the insulin receptor with or without tub gene product. Suchreporter genes may include but are not limited to chloramphenicoltransferase (CAT), luciferase, GUS, growth hormone or placental alkalinephosphatase (SEAP). Alkaline phosphatase or luciferase assays areparticularly useful in the practice of the invention as the enzyme issecreted from the cell and/or is easily assayed. Wherein a NPY promoteris used, the cells are stimulated with nerve growth factor (NGF) for aminimum of six hours. Following exposure of the cells to test compoundand subsequently insulin or IGF-1 the level of reporter gene expressionmay be quantitated to determine the test compound's ability to regulatetub gene product activity. Alkaline phosphatase activity may be assayedfrom tissue culture supernatant. In addition, alkaline phosphatase orluciferase activity may be measured by calorimetric, bioluminescent orchemiluminescent assays such as those described in Brownstein, I. et al.(1994, Biotechniques 17:172-177). Such assays provide a rapid andsimple, detection system for pharmaceutical screening.

[0184] In another embodiment of the present invention, subcellularlocalization of tub gene product can be assayed following exposure to atest compound. As demonstrated by the Applicants, following stimulationof cells with insulin the level of tub gene product found in the nucleusdecreases and levels of tub gene product found in the cytoplasmincrease. As an example of this embodiment, cells engineered to expressgreen fluorescent protein (GFP)-tub may be stimulated with insulin orIGF-1 and exposed to test compound. Thus, following exposure of thecells to the test compound, the levels of tub gene product in thenucleus can be measured to determine the test compound's ability toregulate tub gene product activity and localization and to identifythose compounds which result in the cytoplasmic accumulation of tub geneproduct.

[0185] In yet another embodiment, subcellular localization of tub geneproduct following exposure to test compounds may also be determined bymeasuring the phosphorylation of tub gene product's tyrosine residuesand/or serine/threonine residues. As an example, cells expressinginsulin receptor and tub gene product are serum starved and exposed totest compound and are stimulated with insulin or IGF-1 test compound ina 96-well plate. The cells are lysed and centrifuged to remove thenucleus and cellular debris. The cell lysate is added to a second96-well plate which contains immobilized SH2 containing peptides orimmobilized antiphosphotyrosine antibody. In accordance with the presentinvention, SH2 containing peptides comprise any peptide or protein whichcontains an SH2 binding domain including, but not limited to thefollowing proteins or fragments thereof, PLC gamma, Abl, Lck, Hck, Fgr,BLk, Src, Fyn, Yes and Lyn kinases. To detect cytoplasmic tub geneproduct an anti-tub gene product antibody, tagged with a radioactivelabel, is added. The assay may be performed in a 96-well plate to enablehigh-throughput screening and 96 well-based scintillation countinginstruments may be used for readout.

5.5.2. Cell-Free Assays

[0186] In addition to cell based assays, non-cell based assay systemsmay be used to identify compounds that regulate or alter the activity oftub gene product. In accordance with the invention, recombinantlyexpressed tub gene products, including phosphorylated tub gene products,or cell lysates obtained from cells that express tub gene products maybe used in the screening assays described herein. Such compounds may actas agonists or antagonists of tub gene product activity and may be usedin the treatment of body weight disorders.

[0187] In one embodiment of the cell free assays of the invention, theinteraction of tub gene product with SH2 domains of proteins such as PLCgamma (carboxy terminal SH2), Abl, Lck, Src, etc. following exposure totest compounds. To determine these interactions, a scintillationproximity assay (SPA) may be utilized (SPA kit is provided by AmershamLife Sciences, Illinois). The assay utilizes the SH2 domain of proteinssuch as PLC gamma (the carboxy terminal SH2), Abl, Lck and Src or otherrelevant SH2 domains immobilized on the surface of a 96-well plate. Testcompounds are added which are either agonists or antagonists. Tyrosinephosphorylated tub gene product is added to the assay system. tub geneproduct binding to the immobilized SH2 domains is measured byscintillation proximity assay. The assay may be performed in 96-wellplates to enable high-throughput screening and 96 well-basedscintillation counting instruments such as those manufactured by Wallaceor Packard may be used for readout.

[0188] Alternatively, in yet another embodiment of the cell free assaysof the present invention, activation of tub gene product followingexposure to a test compound may be determined by measuring the tyrosinephosphorylated state of tub gene product. As an example, tub geneproduct may be immobilized to the surface of 96-well plates. Theimmobilized tub gene product is exposed to cellular extracts obtainedfrom cells stimulated with insulin with or without the test compound.Following incubation, the cell extract is removed and the tyrosinephosphorylated state of tub gene product is determined by the ability ofan antibody which recognize phosphorylated tyrosine residues tospecifically bind the immobilized tub gene product. The interactionbetween tub gene product and the antibody may be determined byscintillation proximity assay. Such assays provide high-throughputassays which serve as simple, easily automatable detection systems forpharmaceutical screening.

5.6. Pharmaceutical Preparations and Methods of Administration

[0189] The compounds that are determined to affect tub gene expressionor gene product activity can be administered to a patient attherapeutically effective doses to treat or ameliorate weight disorders,including obesity. A therapeutically effective dose refers to thatamount of the compound sufficient to result in amelioration of symptomsof body weight disorders.

5.6.1. Effective Dose

[0190] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Compounds which exhibit large therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

[0191] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

5.6.2. Formulations an Use

[0192] Pharmaceutical compositions for use in accordance with thepresent invention may be formulated in conventional manner using one ormore physiologically acceptable carriers or excipients.

[0193] Thus, the compounds and their physiologically acceptable saltsand solvates may be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

[0194] For oral administration, the pharmaceutical compositions may takethe form of, for example, tablets or capsules prepared by conventionalmeans with pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

[0195] Preparations for oral administration may be suitably formulatedto give controlled release of the active compound.

[0196] For buccal administration the compositions may take the form oftablets or lozenges formulated in conventional manner.

[0197] For administration by inhalation, the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebuliser, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

[0198] The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

[0199] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

[0200] In addition to the formulations described previously, thecompounds may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the compounds may be formulated with suitable polymeric orhydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

[0201] The compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

6. EXAMPLE Genetic Mapping of the Tub Locus

[0202] In the Example presented herein, studies are described which,first, define the genetic interval within which the tub gene lies, and,second, successfully narrow the interval to approximately 0.25 cM.

6.1. Materials and Methods

[0203] The tubby phenotype. The tubby phenotype was assessed by weighingthe mice. Females weighing less than 35 grams at 150 days wereclassified as normal (i.e., either +/+ or tub/+), while those weighinggreater than 43 grams were typed as tub/tub. Males weighing more than 55grams at 150 days were classified as tub/tub while males weighing lessthan 55 grams were classified as unknown.

[0204] The markers used to genotype the crosses were those identifiedand mapped at the Whitehead Institute at the Massachusetts Institute ofTechnology (Dietrich et al., 1992, Genetics 131:423-447).

[0205] The European backcross mapping panel (Breen et al., 1994, HumanMol. Genet. 3:621-627), which consists of a C57BL/6J×Mus Spretusbackcross, was used to order markers within the tub gene interval.

[0206] Hbb protein polymorphism typing is described in Whitney, J. B.III, 1978, Biochem. Genet. 16:667-672.

[0207] Mouse crosses were performed according to standard procedures.

6.2. Results

[0208] The murine tub gene had previously been mapped to 2.4 cM +/−1.4cM distal of the hemoglobin beta locus (Hbb) on mouse chromosome 7(Jones, J. M. et al., 1992, Genomics 14:197-199). 2.4 cM represents agenetic distance measurement corresponding to 3 observed geneticcrossovers in 125 opportunities. On average, in the mouse genome, thisis equivalent to a physical distance of approximately 4.8 million basepairs. This level of genetic resolution, however, was not satisfactoryfor the cloning of the tub gene. Further, the region of chromosome 7containing the tub gene was not well defined, and no defined markersexisted which flanked the tub locus.

[0209] Described herein, therefore, are genetic crosses which: 1) definethe chromosomal region surrounding the tub gene, and 2) narrow theinterval within which the tub gene is determined to lie to 0.25 cM.

[0210] Specifically, two large crosses segregating the tubby phenotypewere set up and performed, and were typed with available genetic markersknown to map within the relevant region of chromosome 7.

[0211] First, an intercross of [C57BL/6J-tub×DBA/2J] F₁ hybrid mice wasset up. These hybrid mice were the progeny derived from the mating oftwo inbred stains, C57BL/6J-tub/tub and DBA/2J-+/+. In total, 417 F₂progeny, representing 838 independent meioses, were analyzed. Typing allinformative markers against this cross identified a genomic region ofapproximately 4 megabases between the markers D7Mit17 and D7Mit281 whichcontained the tub gene. Two F₂ progeny showed recombination eventsbetween D7Mit17 and the tub locus, thereby establishing this marker asproximal to the tub locus. Five recombinant F₂ progeny demonstrated thatthe tub locus lies proximal to the D7Mit281 marker, thus placing the tublocus between the D7Mit17 and D7Mit281 markers, as shown in FIG. 1. Thedistance between the markers D7Mit17 and D7Mit281 was determined to beabout 2.0 cM, thereby narrowing the interval within which the tub genemust lie to this 2.0 cM region.

[0212] The tub genetic interval was further narrowed by exploiting a byproduct of the way in which tub stock is maintained. tub heterozygotesmust be identified in order to easily maintain the stock because tubhomozygotes have reduced fertility. In order to maintain suchheterozygotes, a C57BL/6J-tub strain was crossed with the congenicC57BL/6J-Hbb^(P) strain. This congenic strain is presumed to begenetically identical to the C57BL/6J strain except for a genomicsegment from a wild mouse strain surrounding and including the Hbblocus. As a result, the C57BL/6J-Hbb^(P) strain has an Hbb allele(Hbb^(P) ) which can be distinguished electrophoretically from theC57BL/6J Hbb allele (Hbb^(S)). Because the Hbb locus is closely linkedto the tub locus, those animals found to be Hbb^(P)/Hbb^(S) werepresumed to be heterozygous at the tub locus as well (a subset ofanimals were tested for the tubby phenotype later, to assure that norecombination between the Hbb and tub loci had taken place).

[0213] Because the two markers under selection for heterozygosity insuch a maintenance scheme are Hbb and tub, the genomic region betweenthese two loci also remains heterozygous as the stock is propagated.However, with each successive generation, this region will narrow, andthe region outside this interval will become homozygous for C57BL/6Jalleles.

[0214] By genotyping of the parental strains C57BL/6J andC57BL/6J-Hbb^(P), the boundaries of the original congenic intervalsurrounding the Hbb locus were established. Proximal of the tub locus,the congenic interval includes the markers D7Mit17, 39, 33, 37 and 38.The congenic interval extends distally beyond the marker D7Mit222 andincludes the markers D7Mitl30 and 53.

[0215] The genotyping of the C57BL/6J-tub/+−Hbb^(S)/Hbb^(P) strainsgenerated herein, led to the finding that the markers D7Mit39, 53 and 22were homozygous for C57BL/6J alleles in each of the animals of thisstrain which were tested. This showed that the congenic interval hadbeen narrowed, through subsequent generations, to an interval betweenD7Mit39 and D7Mit53 (D7Mit39 is 0.3 cM proximal to D7Mit17). Because thetub locus is, by necessity, heterozygous in these animals, it must also,therefore, lie within this D7Mit39-to-D7Mit53 interval. Based on thetyping of 982 progeny of the European backcross mapping panel (Breen etal., 1994, Human Mol. Genet. 3:621-627), this interval was estimated tobe approximately 0.5 cM.

[0216] Next, the tub maintenance stock was used as a cross. Becauseheterozygous mice of this stock (C57BL/6J-tub/+) were heterozygous formarkers within the congenic interval, such a cross represented an F₁intercross segregating tubby in a manner analogous to the tub/DBA/2Jintercross. 394 meioses were genotyped and a single recombinant mousewas identified, demonstrating that the tub locus lies proximal to theD7Mit130 marker. Thus, at this point in the genetic mapping, theproximal boundary of the tub interval was D7Mit17, as defined by therecombinants isolated from the [C57BL/6J-tub×DBA/2J] F₁ intercross andthe distal boundary of the tub interval was D7Mit130, as shown by therecombinant of this C57BL/6J-tub/+intercross. The total number ofmeioses genotyped at this point was 1232: 838 meioses in the[C57BL/6J-tub×DBA/2J] F₁ intercross and 394 meioses in the maintenancestock intercross.

[0217] The size of this region was estimated to be approximately 0.25 cMon the European backcross panel. On average in the mouse genome, such agenetic distance corresponds to a physical distance of approximately 500kb. This finding led to efforts to clone the intervening DNA in anattempt to isolate the tub gene.

7. EXAMPLE Physical Mapping of the Tub Gene Interval

[0218] As a step toward identifying the tub gene, the Example presentedherein describes the physical mapping of the D7Mit17 to D7Mit53 intervalwithin which the tub gene was determined to lie.

7.1. Material and Methods

[0219] Yeast artificial chromosome (YAC) libraries. Two mouse genomicYAC libraries were screened in an effort to identify specific YACscontaining genomic DNA from the tub region. The first YAC library, theWhitehead Mouse YAC Library I, was obtained from Research Genetics(Huntsville, Ala.). The second YAC library, the St. Mary's/ICRF YAClibrary, was a composite library made of YACs constructed at St. Mary'sHospital (London, England) and of YACs constructed at the ImperialCancer Research Fund laboratories and it was obtained from St. Mary'sHospital.

[0220] The YAC libraries were screened by PCR amplification of DNA poolsrepresenting the libraries. A description of a screening protocol can befound in Research Genetics Catalog No. 95020.

[0221] The terminal sequences of the YACs were isolated by vectorettePCR according to Riley et al., 1990, Nucl. Acids Res. 18:2887-2890).Sequencing was performed according to standard procedures.

[0222] YAC ends were mapped according to the protocol described byTuffrey et al., 1993, Hum. Mut. 2:368-374 for single-strandedconformational polymorphism (SSCP) analysis, using SSCPs identifiedbetween C57BL/6J and Mus spretus (the two mouse strains used to generatethe European Backcross mapping panel). Utilizing the YAC end SSCPs itwas possible to determine that the ends of the YACs mapped between theD7Mit17 and D7Mit53 markers.

[0223] P1 bacteriophage. A mouse genomic P1 bacteriophage library(Pierce, J. C. et al., 1992, Mamm. Genome 3:550-558) was screened usingthe Genome Systems screening service. For screening, the ura end of theM72 YAC (M72R) was identified via vectorette PCR (Riley et al., 1990,Nucl. Acids Res. 18:2887-2890). M72R was sequenced and two PCR fragmentswere chosen from this sequence, as shown below:

[0224] M72R-f: 5′-TGC GCA GAA ACA ATC ACC TA-3′ ; (SEQ ID NO:40) and

[0225] M72R-r: 5′-CAA GAC GTG AAC CTG CA-3′ (SEQ ID NO:41) The twoprimers amplify a 129 bp fragment from mouse genomic DNA. The primerswere used by Genome Systems screening service to screen the mousegenomic P1 library.

[0226] Bacterial Artificial Chromosomes (BACs). A MIT/Research mouse BAClibrary obtained from Research Genetics (Catalog No. 96023) was screenedaccording to manufacturer's suggested screening protocol.

7.2. Results

[0227] Described herein are results which describe the physical mappingof the tub region. This region is shown in FIG. 1. In FIG. 1, geneticmarkers are indicated above the top line, while YACs spanning the regionare shown below this. The checkered P1 and BAC clones were analyzed bysequence sampling and exon trapping (see Section 8, below). Overlapsbetween clones were identified by PCR amplification of clones withphysical markers in the region. The tub gene, as described, below, inthis section, was mapped between D7Mit17 and D7Mit53.

[0228] The markers D7Mit 127, 219, 63, 280, 236 and 130 were mappedbetween the D7Mit17 and D7Mit53 markers on the European Backcross panel(Breen et al., 1994, Human Mol. Genet. 3:621-627). These markers,including the D7Mit17 and D7Mit53 markers, were used, therefore, toscreen the MIT YAC library.

[0229] Screening with these markers resulted in the identification of aset of YACs which constituted two contigs. Specifically, the contigaround D7Mit17 included YACs M65, M70 and M72, while the contig aroundD7Mit53 included M49, M79 and M31.

[0230] In order to clone the gap between the two YAC contigs, physicalPCR markers at the ends of the YACs were established, via vectorette PCR(Riley, 1990, Nucl. Acids Res. 18:2887-2890), with which to rescreen theYAC library. The resulting PCR products were sequenced and PCR screeningprimers were chosen. The trp ends of YACs M70 and M31 were isolated (trpends will be referred to herein as the left end of the YACs, e.g., M70L,while the ura ends will be referred to herein as the right ends), andwere genetically mapped, as described, above, in Section 6.1, to the tubregion of mouse chromosome 7 in order to show that they were not derivedfrom chimeric YACs. These ends were then used to screen the St.Mary's/ICRF YAC library.

[0231] One YAC, M84, was identified by both M70L and M31L. Thus, asingle contig spanning the D7Mit17 to D7Mit53 was established. Theminimal contig consisted of M65, M72, M84, M31, M79 and M49, as shown inFIG. 1.

[0232] In order to further aid in gene identification and to confirm theintegrity of the YAC contig described above, P1 bacteriophage andbacterial artificial chromosomes (BACs) were established for theinterval between D7Mit17 and D7Mit130. These P1 clones and BACs overlapto form three contigs separated by two gaps, as shown in FIG. 1.

8. EXAMPLE Identification of a Candidate Tub Gene

[0233] In the Example presented herein, a gene is identified, via exontrapping and sequence sampling, within the cloned DNA described in theExample presented, above, in Section 7, which corresponds to a candidatetub gene. Specifically, Section 8.1 describes the exon trapping andsequencing analyses, while Section 8.2 describes the cloning of putativetub gene cDNA clones.

8.1. Exon Trapping and Sequence Sampling of Tub Gene Interval DNAMaterials and Methods

[0234] Eleven P1 (P1, P2, P3, P4, P6, P7, P8, P10, P11, P13 and P14) andtwelve BAC (B1, B2, B3, B4, B5, B6, B7, B9, B12, B13, B14 AND B15)clones were subcloned into the D-pSPL3, vector, exon trapped andsequence sampled, as described below.

[0235] Exon trapping. The exon trapping analysis was performed usingGibco BRL Exon Trapping System (Cat. No. 18449-017) and using theD-pSPL3 vector, a modified version of the pSPL3 vector (Gibco BRL LifeSciences). In this system, exons are trapped from genomic DNA subclonedinto the vector as a result of the interaction between the vector splicesite and splice sites flanking exons in the genomic DNA.

[0236] D-pSPL3 was derived from the splicing vector pSPL3 (Gibco BRLLife Sciences) by deletion of the NdeI (1119)-NheI (1976) fragment inthe HIV tat intron to eliminate the cryptic splice-donor site atposition 1134 in the pSPL3 sequence. Stocks of BamHI-cut and PstI-cutD-pSPL3 DNA were prepared by digesting 50-100 μg DNA with thecorresponding enzyme and dephosphorylating the linearized vector withcalf intestinal alkaline phosphatase as specified by the manufacturers(New England Biolabs and Boerhinger Mannheim, respectively). Thelinearized vector was purified away from uncut plasmid DNA by agarosegel electrophoresis and electroelution and assayed to assess the levelof uncut and self-ligated vector as described elsewhere (Pulido andDuyk, 1994, in Current Protocols in Human Genetics, Wiley Pub., pp2.2.1-2.3.1).

[0237] Briefly, P1 and BAC clone DNA was prepared from overnightcultures (100 ml LB/kanamycin 25 μg/ml) by standard alkaline lysis,treated with RNase A, purified by phenol/chloroform/isoamyl alcohol(25:24:1) extraction, ethanol precipitated, rinsed in 70% ethanol, driedand resuspended in 400 μl deionized water. 5-10 μg P1/BAC DNA was cutwith either BamHI and BglII, or PstI, as specified by the manufacturer(New England Biolabs, Beverly, Mass.). The digested DNA was phenolextracted, ethanol precipitated and resuspended in 50 μl deionizedwater.

[0238] Exon trapping was then completed as described in the Gibco BRLExon Trapping Manual. Briefly, the D-pSPL3 clones were transfected intoCOS-7 cells. RNA was isolated and first strand cDNA was synthesized. Tworounds of nested PCR specifically amplified transcripts derived from theD-pSPL3 clones. These PCR products were cloned into the vector pAMP10.Clones from this pAMP10 library of trapped fragments were then analyzedby PCR to determine insert sizes. Clones with insert sizes greater than150 bp were sequenced using M13 forward and reverse primers. One of theD-pSPL3 subclones was designated ium008p004, and was sequenced.

[0239] A 90 bp fragment, designated P8X1, was PCR amplified using thesequence of this subclone insert. The P8X1 fragment was generated usingtwo PCR primers which were designed using the ium008p004 sequence asfollows:

[0240] P8X1F1: 5′-GCG GAT ACA GAC TCT CTC AT-3′ (SEQ ID NO:42)

[0241] P8X1R1: 5′-GAG GAC AAA TGT CCT AGG CT-3′ (SEQ ID NO:43)

[0242] The 90 bp P8X1 DNA fragment was PCR amplified from first strandcDNA made from C57BL/6J mouse brain RNA. Standard cDNA synthesis and PCRprocedures were utilized.

[0243] Sequence sampling. Sequence sampling is a technique for rapidlydetermining whether coding sequences were present in a nucleic acidsample of interest (See Claverie, J. M., 1994, Genomics 23:575-581). Theinserts in D-pSPL3 clones described above were sequenced in bothorientations using the following primers:

[0244] SPL3A: 5′-CAT GCT CCT TGG GAT GT-3′ (SEQ ID NO:44)

[0245] SPL3C: 5′-TGA GGA TTG CTT AAA GA-3′ (SEQ ID NO:45) After vectortrimming and quality assessment, the resulting sequences were comparedto nucleic acid and protein databases using BLAST algorithms (Altschul,S. F. et al., 1990, J. Mol. Biol. 215:403-410).

Results

[0246] In order to look for genes within the cloned DNA, described,above, in Section 7, within the interval containing the tub gene, P1 andBAC clones were subcloned into the D-pSPL3 vector and exon trapped andsample sequenced, as described, above, in the Materials and Methodsportion of this section. One of the D-pSPL3 subclones, designatedium008p004, was derived from a D-pSPL3 library made from the P8 clone(see FIG. 1). A 327 base pair portion of the P1 insert in ium008p004 wassequenced. The protein sequence encoded by this portion of ium008p004showed homology to two translated sequences in the GenBank nucleic aciddatabase. Two primers were selected from the region of homology and usedto amplify a DNA fragment of 90 bp, called P8X1, having the followingsequence:

[0247] 5′GAGACAAATG TCCTAGGCTT CAAGGGACCT CGGAAGATGA GTGTGATCGTCCCAGGCATG AACATGGTTC ATGAGAGAGT CTGTATCCGC 3′ (SEQ ID NO:46)

[0248] The ium008p004 homologies were to Genbank sequences Z48334 andX69827. Z48334 is the partial sequence of a Caenorhabditis eleganscosmid, F10B5. One of the putative genes identified within this sequencecontains a 425 amino acid open reading frame, designated F10B5.4 (WilsonR. et al, 1994, Nature 368:32-38). X69827 is a mouse 981 bp partial cDNAwith a potential open reading frame of 323 amino acids. This sequencehas been shown to have similarity to the family of phosphodiesteraseproteins (Vambutas, V. and Wolgemuth, D. J., 1994, Biochim. Biophys.Acta. 1217:203-206).

[0249] The above sequence was flanked by consensus splice sites, furtherdemonstrating that the sequence is from an exon, or a coding region, ofa gene. The homology to a known gene, as described above, coupled withthe presence of consensus splice sites strongly suggested that thisregion of the ium008p004 clone corresponded to a portion of the codingregion of a gene. Given its location within the interval in which thetub gene is located, this putative gene, which was designated CBT9,represented a tub gene candidate.

8.2. Isolation of CBT9 cDNA Clones Materials and Methods

[0250] cDNA cloning. In order to isolate a longer cDNA of the CBT9 gene,the P8X1 fragment was used as a probe to screen a Stratagene (La Jolla,Calif.) mouse brain cDNA library (#936309). For hybridization, AmershamRapid Hyb Buffer (Cat. No. RPN1639) was utilized according tomanufacturer's protocol. A final washing stringency of was 2×SSC/0.1%SDS at 65° C. was attained and autoradiography was performed overnight.One million clones were screened. Among the clones identified was thefume009 clone, a 1.15 kb cDNA, which was then sequenced.

[0251] The fume009 clone was used to screen a mouse hypothalamus cDNAlibrary. This library was constructed from poly-A⁺ RNA from 6 week oldC57BL/6J mice. First and second strand cDNA was made from the poly-A⁺RNA using standard procedures. cDNA was ligated into Uni-ZAP XR lambdavector and packaged using a Stratagene kit (Cat. No. 237611). Identicalwashing conditions as described above were utilized. The screenidentified a 6.0 kb clone, designated fumh019, which was sequenced. Thefumh019 cDNA clone contains the entire CBT9 gene coding region. The CBT9sequence is further discussed, below, in the Example presented inSection 12.

Results

[0252] In order to isolate CBT9 cDNA clone, the P8X1 fragment was used,as described, above, in the Materials and Methods portion of thissection, to screen a mouse brain cDNA library. This screen resulted inthe isolation of the fume009 1.15 kb cDNA clone.

[0253] The fume009 cDNA clone was then used, as described, above, in theMaterials and Methods portion of this section, to screen a mousehypothalamus cDNA library. This screen resulted in the isolation of a6.0 kb cDNA clone, designated fumh019.

[0254] The fumh019 cDNA clone was sequenced and was determined tocontain the entire CBT9 coding region. The CBT9 nucleotide and aminoacid sequence are described, below, in the Example presented, below, inSection 12.

9. EXAMPLE Characterization of the Expression of the CBT9 Gene

[0255] In the Example presented herein, Northern analysis data isdescribed which characterizes the CBT9 gene (see Section 8, above).Specifically, experiments are presented herein which evaluate theexpression of CBT9 in a number of mouse tissues obtained from wild typeand tub mice. The results presented herein are consistent with the CBT9gene being the tub gene.

9.1. Material and Methods

[0256] Northern analysis. The P8X1 DNA fragment and the fume009 cDNAclone were used to probe Northern blots containing total mouse RNA.

[0257] Total RNA from tub and wild type (C57BL/6J) mice was isolated andutilized for the Northern analysis. All mice were sacrificed by carbondioxide asphyxiation. Tissues were dissected on ice, snap-frozen inliquid nitrogen and stored at −80° C. Total RNA was isolated usingRNazolB (TelTest, Inc.) The total RNA samples were resuspended inRNase-free DEPC-treated water and quantitated by optical densitymeasurement.

[0258] For the Northern blots, 10 μg total RNA of each sample was loadedonto a formaldehyde gel. The gel was blotted onto a nylon membrane usingstandard Northern transfer techniques. The blot was hybridized with P8X1which had been radiolabelled by random priming using a Gibco-BRL kit(Cat. No. 18187-013) according the manufacturer's recommended protocol.For hybridization, Amersham Rapid Hyb Buffer (Cat. No. RPN1639) wasutilized according to manufacturer's protocol. A final washingstringency of 0.1×SSC/0.1% SDS at 65° C. was attained, andautoradiography was performed overnight.

[0259] The Northern blot depicted in FIG. 2 was loaded as follows: lane1, wild type brain without hypothalamus; lane 2, tub brain withouthypothalamus; lane 3, wild type hypothalamus; lane 4, tub hypothalamus;lane 5, wild type heart; lane 6, tub heart; lane 7, wild type lung; lane8, tub lung; lane 9, wild type liver; lane 10, tub liver; lane 11, wildtype kidney; lane 12, tub kidney; lane 13, wild type spleen; lane 14,tub spleen; lane 15, wild type stomach; lane 16, tub stomach; lane 17,wild type muscle; lane 18, tub muscle; lane 19, wild type fat; lane 20,tub fat; lane 21, wild type testis; lane 22, tub testis; lane 23, RNAmolecular weight standards, the sizes of which are indicated by thelines at the right hand side of the blot. Specifically, the sizes are9.49 kb, 7.46 kb, 4.40 kb, 2.37 kb, 1.35 kb and 0.24 kb. The crossesindicate the positions of the 28S and 18S ribosomal RNA molecules. “Wildtype” refers to C57BL/6J mice.

[0260] The Northern blot depicted in FIG. 3 was loaded as follows: lane1, RNA molecular weight standards, the sizes of which are indicated bythe lines at the side of the blot (specifically, such sizes are 9.49 kb,7.46 kb, 4.40 kb, 2.37 kb, 1.35 kb and 0.24 kb); lane 2, wild type brainwithout hypothalamus; lane 3, tub brain without hypothalamus; lane 4,wild type hypothalamus; lane 5, tub hypothalamus; lane 6, wild typeheart; lane 7, tub heart; lane 8, wild type lung; lane 9, tub lung; lane10, wild type liver; lane 11, tub liver; lane 12, wild type kidney; lane13, tub kidney; lane 14, wild type spleen; lane 15, tub spleen; lane 16,wild type stomach; lane 17, tub stomach; lane 18, wild type muscle; lane19, tub muscle; lane 20, wild type fat; lane 21, tub fat; lane 22, wildtype testis; lane 23, tub testis. The crosses indicate the positions ofthe 28S and 18S ribosomal RNA molecules. “Wild type” refers to C57BL/6Jmice.

9.2. Results

[0261] As shown in FIG. 2, a CBT9 transcript of approximately 7.0 kb ispresent in the hypothalamus without brain (lane 2) and in thehypothalamus (lane 4) RNA samples derived from the wild type C57BL/6Jmice as detected by the P8X1 probe. No CBT9 transcript is detectable inother total RNA samples derived from wild type mouse tissues.

[0262] As is further shown in FIG. 2, a CBT9 transcript of approximately7.5 kb, i.e., approximately 0.5 kb larger than the transcript seen inthe wild type tissues, is present in both the brain without hypothalamus(lane 3) and hypothalamus (lane 5) RNA samples derived from the tub miceas detected by the P8X1 probe. No CBT9 transcript is detectable in othersamples of total RNA derived from tub mouse tissues. It shouldadditionally be noted that the abundance of the transcript detected bythe P8X1 probe in tub RNA samples is approximately 5-fold greater thanit is in RNA samples from wild type (C57BL/6J) mice.

[0263] In addition, the fume009 clone was used as a probe to verify theresults, described above, which were obtained using the P8X1 fragment asa probe. As shown in FIG. 3, Northern analysis using such a fume009sequence to probe total RNA from tub and wild type mouse tissue samplesyielded the same CBT9 results which were observed using the P8X1 probe.Specifically, a transcript of the same increased size was seen in thetotal RNA samples derived from tub homozygous mice and the same upregulation was observed in the amount of tub RNA present in total RNAsamples derived from tub homozygous animals relative to wild typeanimals.

[0264] A Northern blot analysis of total RNA derived from an animalgenotypically shown to be heterozygous for the tub mutation revealed, asexpected from the above results, the presence of both the 7.5 kb and 7.0kb transcripts in total brain RNA. In addition, a moderate up regulation(approximately two-fold) of CBT9 transcript levels relative to CBT9levels in wild type animals, was observed.

[0265] The results of these Northern analyses strongly suggest that adefect within the CBT9 gene results in the tubby phenotype.Specifically, the difference in size observed between the CBT9transcript in wild type and in tub RNA is consistent with a mutationresulting in the inclusion of exogenous nucleic acid into the tub mRNA.Second, the approximately 5-fold up regulation of CBT9 RNA levels in theRNA samples derived from the tub/tub homozygotes relative to levelsobserved in RNA samples derived from the wild type mice suggests thatsuch high levels of this transcript are related to the obesity phenotypeseen in tubby animals. This may be the result of a negative feedbackloop induced by the absence or malfunction of the protein encoded by themutant tub (CBT9) gene. Third, in total mouse RNA, the CBT9 gene isexpressed in the brain, including the hypothalamus, a region of thebrain which is known to be involved in the control of body weight (Bray,G. A., 1992, Progress in Brain Res. 93:333-341). Finally, the moderateup regulation seen in the heterozygous animals is consistent with therecessive inheritance pattern of the tubby phenotype, in whichheterozygotes are not obese, but, nonetheless, have been shown toexhibit some phenotypic differences relative to homozygous wild typecontrol animals (Nishina, P. M. et al., 1994, Metabolism 43:554-558).

10. EXAMPLE CBT9 Southern Blot Analysis

[0266] In the Example presented herein, the results of a Southern blotanalysis are described which indicate that homologs of the murine CBT9gene are present and have been conserved in other mammalian species.

10.1. Material and Methods

[0267] Southern blot analysis. Two PCR primers were designed from theCBT9 nucleotide coding sequence, as follows:

[0268] P8X9F1: 5′-GGA CAA GAA GGG GAT GGA C-3′ (SEQ ID NO:47)

[0269] P8X10R1: 5′-CCG TGG ATG ATC TGG AAG T-3′ (SEQ ID NO:48)

[0270] The primers were used to amplify, via RT-PCR, a 650 bp cDNAfragment (designated P8X9-10) from C57BL/6J mouse whole brain RNA.Standard RT-PCR conditions were utilized. The band was gel-purified andrandom-prime radiolabelled, as described above.

[0271] The resulting probe was hybridized to a Southern blot ofEcoRI-digested genomic DNA (BIOS Laboratories; #EBM-100E) from variousmammals. Each lane was loaded with 8 μg of digested genomic DNA. Forhybridization, Amersham Rapid Hyb Buffer (Cat. No. RPN1639) was utilizedaccording to manufacturer's protocol. A final washing stringency of0.5×SSC/0.1% SDS at 65° C. was attained, and blots were exposedovernight with an intensifying screen at −80° C.

[0272] The lanes of the Southern blot depicted in FIG. 5 were loaded asfollows: lane 1, markers: lambda DNA digested with HindIII (band sizesare as indicated in the figure); lane 2, mouse; lane 3, hamster; lane 4,rat; lane 5, rabbit; lane 6, dog; lane 7, cat; lane 8, cow; lane 9,sheep; lane 10, pig; lane 11, marmoset; lane 12, human.

10.2. Results

[0273] A Southern blot analysis was conducted using a CBT9 probe(P8X9-10; see Section 10.1 for details) and a DNA blot containingEcoRI-digested mammalian genomic DNA of various species, as describedabove, in Section 10.1. As is shown in FIG. 5, the CBT9 probe detectshomologous sequences in each of the mammalian DNA sample represented onthe blot. This result provides additional evidence that the CBT9sequence used as a probe is part of a gene and, additionally,demonstrates that the sequences show a high level of conservation amonga wide range of mammalian species.

11. EXAMPLE CBT9 In Situ Hybridization Analysis

[0274] In the Example presented herein, the results of an in situhybridization analysis are described which verify that the CBT9 gene isexpressed in the brain. Primary CBT9 gene expression occurred within thehippocampus, hypothalamus and cortex. Weaker hybridization could be seenthroughout the brain.

11.1. Material and Methods

[0275] In situ Hybridization Localization: Brains from 6 month-old C57BL/6J mice were removed flash frozen at −80° C. and stored at −80° C.until use. 10 μm frozen sections of brains were post-fixed with 4%PFA/PBS for 15 minutes. After washing with PBS, sections were digestedwith 1 μg/ml proteinase K at 37° C. for 15 minutes, and again incubatedwith 4% PFA/PBS for 10 minutes. Sections were then washed with PBS,incubated with 0.2 N HCl for 10 minutes, washed with PBS, incubated with0.25% acetic anhydride/1 M triethanolamine for 10 minutes, washed withPBS and dehydrated with 70% ethanol and 100% ethanol. Hybridizationswere performed with ³⁵S-radiolabelled (5×10⁷ cpm/ml) cRNA probesencoding a 1.15 kb segment of the coding region of the mouse clonefume009 in the presence of 50% formamide, 10% dextran sulfate, 1×Denhardt's solution, 600 mM NaCl, 10 mM DTT, 0.25% SDS and 100 μg/mltRNA for 18 hours at 55° C. After hybridization, slides were washed with5×SSC at 55° C., 50% formamide/2×SSC at 55° C. for 30 minutes, 10 mMTris-HCl(pH 7.6)/500 mM NaCl/1 mM EDTA (TNE) at 37° C. for 10 minutes,incubated in 10 μg/ml RNase A in TNE at 37° C. for 30 minutes, washed inTNE at 37° C. for 10 minutes, incubated once in 2×SSC at 50° C. for 30minutes, twice in 0.2×SSC at 50° C for 30 minutes, and dehydrated with70% ethanol and 100% ethanol. Localization of mRNA transcripts wasdetected by film emulsion autoradiography followed by dipping slides inphoto-emulsion for precise autoradiographic localization.

11.2. Results

[0276] The fume009 cDNA clone was used as a probe for an in situhybridization analysis. Specifically, the 1.15 kb fume009 probe washybridized to sections of wild type C57BL/6J mice. As shown in FIG. 5,the CBT9 transcript is expressed in the hypothalamus and other regionsof the brain, consistent with the above-described Northern analysisdata, which was presented in Section 9, above.

[0277] Specifically, an mRNA transcript hybridizing to the 1.15 kBfume009 antisense cRNA probe was localized to discrete regions of thebrain of both C57BL/6J wild type mice (FIG. 5) and tub homozygous mice.Signal was observed in the hypothalamus adjacent to the 3rd ventricle intwo “nuclear bodies” (indicated by dense clustering of nuclei) as wellas at the base of the hypothalamus adjacent to the optic chiasm in thetissue from both mice. Thus, expression in the hypothalamus is highestin the paraventricular, ventromedial and arcuate nuclei.

[0278] In addition, signal was detected in scattered cells in thesubcortical temporal lobe and in hippocampus in the tissue sections fromboth mice. FIG. 5 shows the regions of localization of tub genetranscript in the brain of C57BL/6J mice (the arrows indicate thoseregions where signal was detected). Weaker hybridization was observedthroughout the brain. No distinct signal was observed in heart, spleen,liver, lung, skeletal muscle, pancreas, small intestine and stomach ofeither the C57 BL/6J wild type mice or tub homozygous mice.

12. EXAMPLE Identification of CBT9 as the Tub Gene

[0279] Presented in this Example is, first, a mutational analysis of theCBT9 gene, which compares CBT9 gene sequences within nucleic acidderived from wild type and tub animals. Specifically, a CBT9 splice sitemutation is identified within tub genomic DNA which is absent from wildtype genomic DNA. Second, the nucleotide and derived amino acid sequenceof the CBT9 gene is presented. The results disclosed herein, coupledwith the results presented, above, in Sections 6 to 11, identify theCBT9 gene to be the tub gene.

12.1. Materials and Methods

[0280] PCR analysis. A number of primers were designed to amplify theentire open reading frame of CBT9 from tub and wild type mice in orderto identify the location of the mutation in the tub gene. The followingtwo primers amplified different sized cDNA fragments when amplifyingtub-derived versus wild type-derived nucleic acid:

[0281] PX1R: 5′-TGA GAC AAA TGT CCT AGG CT-3′ (SEQ ID NO:49)(corresponding to CBT9 base pair 1113 to 1132);

[0282] PX12R: 5′-TGG ACA GAG CAA TGG CGA AG-3′ (SEQ ID NO:50)(corresponding to CBT9 base pair 1489 to 1470)

[0283] Standard PCR conditions and sequencing procedures were utilized.

12.2. Results 12.2.1. CBT9 Mutational Analysis

[0284] In order to more definitively show that the CBT9 genecorresponded to the tub gene, a PCR study was conducted to define themutation causing the CBT9 transcript size change observed in tub micerelative to the CBT9 transcript size observed in RNA of wild type mice.Of the PCR primer pairs utilized, only one resulted in a sizedifferential between the fragment amplified using tub-derived nucleicacid and the fragment amplified using wild type-derived nucleic acid(see Section 10.1 for details).

[0285] Specifically, utilizing this primer pair (i.e., PX1R and PX12R) acDNA was amplified from wild type (C57BL/6J) brain RNA which was about350 bp, while a cDNA fragment was amplified from tub brain RNA which wasabout 800 bp. The amplification of both wild type (C57BL/6J) and tubgenomic DNA resulted in a band of approximately 900 bp.

[0286] It should be noted that the size differential, approximately 450bp, between the tub and wild type cDNA amplified fragments roughlycorresponds to the difference in transcript size (i.e., 7 vs. 7.5 kb)observed between tub and wild type RNA in the CBT9 Northern analysisdescribed, above, in the Example presented in Section 9. By sequencing(see below) it was determined that the precise size difference betweenthe tub and wild type cDNA amplified sequences was 398 bp.

[0287] The 900 bp fragment amplified from genomic DNA reveals thepresence of a second intron within the amplified region. Only one ofthese introns (of approximately 100 bp in length) was processedcorrectly in the tub animals, as discussed below.

[0288] The cDNA and genomic amplified fragments in the region of themutation were sequenced and the wild type- and tub-derived sequenceswere aligned, as shown in FIG. 7A-7D. For orientation of the genomicsequence depicted in FIG. 7A-7D with the full length CBT9 cDNA codingsequence shown in FIG. 6A-6D, bases 1-12 and 411-437 in FIG. 7A-7Dcorrespond to bases 1373-1384 and 1385-1411 of FIG. 6A-6D. In FIG.7A-7D, the two top sequences are from genomic DNA derived from tub andwild type C57BL/6J mice, as indicated. The bottom two sequences arederived from cDNA from tub and wild type mice, as indicated. Thevertical arrow shows the position of the tub mutation. The horizontalbox indicates the consensus splice site sequence in C57BL/6J which isabolished in the tub genomic DNA. The asterisks indicate the intronwhich is erroneously not spliced out of the mature tub mRNA.

[0289] The portion of the CBT9 gene sequence in FIG. 7A-7D depicts onlythe genomic region near the mutation site. This alignment revealed asingle base pair difference of a G to T transversion in the first baseof the splice site (GTGACT; see boxed region of FIG. 1) of the intronbetween base 1384 and 1385 of the open reading frame of the genomic DNA.This mutation abolishes the splice site, resulting in retention of anintron of approximately 450 bp in the amplified cDNA derived from thetub RNA. To confirm that the identified sequence change did not simplyrepresent a polymorphism, the splice site was sequenced in 32 additionalmouse strains. In each of the strains, the DNA sequence at the putativemutation site was identical to that observed in the wild type C57BL/6Jstrain.

[0290]FIG. 8 depicts a schematic representation of the splicing defectwithin the CBT9 in tub mice. The top half of the figure shows thenormal, wild type splicing of the intron from C57BL/6J RNA and thepredicted carboxy terminus of the wild type CBT9 protein. The G to Tmutation of the first base of the intron within the CBT9 gene in tubmice abolishes splicing of this intron, causing the intron to beretained within the mature mRNA. The predicted tub mutant CBT9 protein,therefore, is abnormal. Specifically, due to translation of intronicsequence, this mutant tub gene product lacks the final 44 amino acidresidues of the normal CBT9 protein and, instead, contains 24intron-encoded amino acid residues at its carboxy terminus which areerroneously added to the tub protein until a stop codon within theintronic sequence is reached.

12.2.2. CBT9 Nucleotide and Amino Acid Sequence

[0291] As discussed in Section 8.2, above, the fumh019 CBT9 cDNA clonewas sequenced. Sequencing revealed that the fumh019 cDNA clone containedthe entire CBT9 open reading frame.

[0292] The nucleotide sequence and amino acid sequence of CBT9 is shownin FIG. 6A-6D. The CBT9-encoded protein is 505 amino acid residues inlength. CBT9 is a novel gene, with no identical sequences present inpublished databases.

[0293] The entire CBT9 coding region of the Mus spretus and A/J mousestrains were sequenced and no non-conservative amino acid changes ineither strain as compared to the C57BL/6J tub sequence were found.

[0294] The CBT9 gene product is a hydrophilic protein, with an estimatedpI of 9.2, which lacks any obvious secretary sequence, mitochondrialtransit peptide or transmembrane domain. The gene product contains aregion consisting of two runs of serine amino acid residues separated byeight acidic amino acid residues (amino acid residues 191-211), whichcould serve as a hinge between domains of the protein. In addition, twopotential dibasic protease cleavage sites are present at amino acidpositions 302 and 383, and two potential glycosylation sites are presentat amino acid positions 205 and 426.

[0295] The carboxy half of the CBT9 gene product shows similarity toseveral sequences in the public protein databases and/or encoded bysequences present in public nucleotide databases, including p4-6, amouse testis cDNA (Genbank X69827); F10B5.4 (Genbank Z48334), a C.elegans genomic sequence; DM87D3S (Genbank Z50688) a Drosophila STS; andys86c04.r1 (Genbank H92408), a human retinal cDNA; as well as severalrice, maize and Arabidopsis ESTs. With the exception of the mouse testiscDNA p4-6, none of these sequences has been functionally characterized.p4-6 was isolated by screening of a cDNA library with a ratphosphodiesterase probe (Vambutas, V. & Wolgemuth, D. J. 1994, Biochim.Biophys. Acta 1217: 203-206).

[0296] Upon alignment of the CBT9 gene product and the sequences showingsimilarity to CBT9, certain regions were shown to be completelyconserved. Specifically, the two dibasic protease cleavage amino acidresidues and the cysteine amino acid at the penultimate CBT9 positionare all completely conserved among all the CBT9-related sequences.

[0297] The data presented in Sections 6 to 11 above, including mappingdata, and Northern and in situ analyses, and the mutational analysisdata presented in this Section demonstrating that the tubby phenotype isassociated with a splicing defect within the CBT9 gene which results ina major alteration of the carboxy terminus of the CBT9 gene product,represent conclusive evidence that the CBT9 gene is the tub gene.Specifically, CBT9 maps within the 0.25cM interval that the tub gene hasbeen shown, herein, to map. Further, the CBT9 gene is expressed in thebrain, including the hypothalamus, a region known to be involved in bodyweight control. Additionally, the CBT9 transcript in tub animals islarger than the CBT9 transcript found in wild type C57BL/6J animals andit has been shown herein that this increase in size is due to a singlebase mutation in a CBT9 splice site which results in the incorrectsplicing of the RNA such that a 398 nucleotide intron remains within themature mRNA. As a result, the protein which is translated from such amutant transcript exhibits an abnormal carboxy terminus. Presumably as aresult of this defect, the CBT9 mRNA is upregulated approximately 5-foldin homozygous tub/tub mice. The heterozygous tub/+ mice showed a moremodest upregulation, as would be expected, given the heterozygous tubphenotype. In summary, therefore, the CBT9 gene has successfully beenidentified to be the tub gene.

13. EXAMPLE Cloning and Characterization of the Human Tub Gene

[0298] The Example presented herein describes the successful cloning andcharacterization of the human tub gene, which is involved in the controlof human body weight. Both the human tub gene and gene product exhibit astriking level of similarity to the murine tub gene and gene product.

13.1. Materials and Methods

[0299] P8X5-1 tub probe generation: The 950 base pair P8X5-1 tub genecDNA probe was generated by standard PCR amplification of the murinecDNA clone fumh019, described, above, in Section 8. The followingprimers were utilized for the amplification:

[0300] P8X5R1: 5′-CCG ACT CGA TTG CCA GTG TA -3′ (SEQ ID NO:51)

[0301] P8X1F1: 5′-GCG GAT ACA GAC TCT CTC AT -3′ (SEQ ID NO:52)

[0302] Upon amplification, the probe was gel purified and radiolabelledaccording to standard protocols.

[0303] cDNA screening: Screening was performed on a human fetal braincDNA library (Clontech #HL1149x). Hybridization was performed for 4hours at 65° C. using Amersham Rapid Hyb buffer (Cat. # RPN1639)according to the manufacturer's protocol. A final washing stringency of1.0×SSC/ 0.1% SDS at 50° C. for 20 minutes was achieved. Autoradiographywas performed overnight.

[0304] DNA sequencing: Standard DNA sequencing techniques were utilizedfor the sequencing of the resulting putative human tub cDNA clones.

13.2. Results

[0305] The 950 base pair P8X5-1 murine tub gene probe, described, above,in Section 13.1, was used to screen a human fetal brain cDNA library forclones corresponding to the human tub gene. Screening conditions were asdescribed, above, in Section 13.1.

[0306] Screening of the human cDNA library yielded thirteen independentpositive clones. Among these clones were those designated CBT9H1, CBT9H2and CBT9H3, which have been deposited with the ATCC. Sequencing revealedthat the entire coding region of the human tub gene was contained withinthese partially overlapping clones.

[0307] The nucleotide and derived amino acid sequences of the human tubgene are shown in FIG. 9A-9D. As shown in FIG. 9A-9D, the human tub geneencodes a 506 amino acid protein. The human tub gene product encodes ahydrophilic protein exhibiting an estimated pI of 9.2 which lacks anyobvious secretory sequence, mitochondrial transit peptide ortransmembrane domain. The gene product contains a region consisting oftwo runs of serine amino acid residues separated by a acidic amino acidresidues (amino acid 191-211) which could serve as a hinge betweendomains of the protein. In addition, there are two potential dibasicprotease cleavage sites at amino acid positions 301-306 and 381-384, aswell as two potential N-glycosylation sites at amino acid residues 206and 427.

[0308] The human tub gene and gene product exhibit a striking similarityto the murine tub gene and gene product. Specifically, the human tubgene is 89% identical, at the nucleotide level, to the murine tub gene.Further, the 506 amino acid human tub gene product exhibits a 94%identity to the 505 amino acid murine tub gene product. Amino acidresidue 201 represents the only amino acid insertion between the two tubgene product sequences. Specifically, the human tub amino acid residue201 corresponds to an insertion between murine amino acid residues 200and 201. The carboxy half of the tub gene product is particularly highlyconserved. The final 260 amino acid residues of the human and mouse tubgene products differ by only a single residue. Specifically, murine tubgene product amino acid residue 399 is a cysteine, while thecorresponding human tub gene product amino acid residue 400 is serine.

[0309] In summary, the results presented herein represent the successfulcloning of the human tub gene.

14. EXAMPLE Human and Murine Tub Gene Alternative Splicing

[0310] The Example presented herein describes the discovery that boththe human and murine tub genes produce alternatively splicedtranscripts. Specifically, it is shown that tub transcripts are producedwhich either contain or are lacking the sequence corresponding to tubexon 5. Quantitative variation between the relative amounts ofalternatively spliced species produced is also described.

14.1. Material and Methods

[0311] RT-PCR. First strand cDNA was synthesized from total RNA usingSuperScript (Gibco-BRL) according to supplier's protocol. Subsequent PCRconditions were as follows: Hot start with 0.5U AmpliTaq, followed by 30cycles at 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for 1minute. Products were electrophoresed on 2% agarose gels. RT-PCRproducts to be sequenced were run on LMP agarose, excised, digested withβ-Agarase (New England Biolabs), precipitated and resuspended in water.The same conditions were utilized for amplification of both human andmouse RNA populations.

[0312] The primers utilized for mouse sequence amplification werederived from murine tub exons 4 and 6: P8X5R: 5′-CCG ACT CGA TTG CCA GTGTA-3′; (SEQ ID NO:53) and CBT9R5: 5′-GGA GCT GTT TTC ATC CTC ATC-3′ (SEQID NO:54).

[0313] The primers utilized for human sequence amplification werederived from human tub exons 4 and 6: hCBT9F11: 5′- GAA GGA GAA GAA GGGAAA GC-3′; (SEQ ID NO:55) and hCBT9R11: 5′-GGG TGT TAC TAT TTA GCT GG-3′(SEQ ID NO:56)

[0314] Other techniques. All other techniques were performed accordingto standard procedures and/or as described in the Examples presentedabove. Primers used for genomic PCR amplification were derived from tubexons 4 and 5: X4F1: 5′-TTC AAG AGG CCG ACT CGA TT -3′; (SEQ ID NO:57)and X5R1: 5′-TTC CTC TGC ATC GTG GCA C-3′ (SEQ ID NO:58)

14.2. Results

[0315] RT-PCR from mouse brain RNA using primers derived from exons 4and 6, as described in Section 14.1, above, resulted in theamplification of two products. Sequencing of these products showed thatthey differ by the presence or absence of sequence corresponding to exon5. RT-PCR of RNA from C57BL/6J mice consistently yielded more of theamplified product containing exon 5. This result was shown to be truefor 7 other strains tested.

[0316] RT-PCR performed using brain RNA derived from the Mus spretusstrain, however, invariably showed a greater abundance of the productlacking exon 5. This was demonstrated in 6 independent M. spretus mice.This quantitative pattern was also found to be exhibited in M.castaneous mice. Genomic PCR revealed that the intron preceding exon 5was 0.5 kb shorter in both M. spretus and M. castaneous strains.Sequencing of a portion of this intron showed that its donor, acceptorand branch point sequences were not affected by the sequence missing inthese strains.

[0317] RT-PCR of total human RNA from several tissues was performed withtwo primers from exons 4 and 6 using the same conditions as for mouseRNA. The amplification primers were hCBT9F11 and hCBT9R11, as described,above, in Section 14.1. Amplification produced two amplified fragmentsof 281 bp and 113 bp. Sequencing revealed that the larger bandrepresented a transcript containing exon 5, while the smaller fragmentwas missing the sequence corresponding to exon 5. Thus, the human tubgene, also exhibits alternate splicing of exon 5. Both the human andmouse exon 5 is 168 base pairs long. Because this length is a multipleof 3, the reading frame of the transcripts lacking exon 5 is conserved.

[0318] It is possible that variant splicing may result in proteins withqualitatively or quantitatively distinct activities. The differentialregulation of alternate splicing may result in individuals withdifferential susceptibilities to obesity. For example, in place of theconstitutive obesity associated with the tub mutation, alleles whichyield a higher amount of protein encoded by transcripts lacking exon 5relative to the level encoded by transcripts containing exon 5 mayconfer a greater susceptibility to obesity only in the context of aparticular environmental and genetic background.

15. EXAMPLE Recombinant Expression of Tub Gene Products

[0319] The Example presented in this Section describes the recombinantexpression of murine and human tub gene products.

15.1. Materials and Methods

[0320] Bacterial Expression

[0321] Murine tub subcloning. cDNA sequence containing the entire murinetub coding region was subcloned into bacterial expression vector pET29*.pET29* is a modified pET29a vector (Novagen, Inc., Madison Wis.)containing an altered Shine-Dalgarno sequence for optimal initiation oftranslation (Chen, H. et al., 1994, Nuc. Acids Res. 22:4953-4957).

[0322] In order to subclone the tub coding sequence into the pET29*vector, site directed mutagenesis was performed on an existing tub cDNAto create a tub sequence with appropriate restriction sites.Specifically, single stranded DNA was rescued from CJ 236 E. colitransformed with pMal-c2 (New England Biolabs, Beverley MA) plasmidcontaining a PCR-derived tub cDNA by infection with K07 M13 helperphage. The single stranded DNA was used as a template for site directedmutagenesis which yielded amplified tub fragments containing alteredends (Kunkel, T. A., 1985, Proc. Natl. Acad. Sci. USA 82:488-491). The5′ end of the amplified fragment was altered such that the tubinitiation codon was contained within an NdeI site (i.e., CATATG), whilethe 3′ end was altered such that part of the tub termination codon wascontained within an EcoRI site (i.e., TGAATTC). The resulting tub cDNAwas excised as a 5′ NdeI to 3′ EcoRI fragment and ligated intoNdeI/EcoRI-digested pET29* vector, to yield the murine pET29*-tubexpression construct.

[0323] In order to produce the murine tub-HIS₆ expression construct,codons for six histidine residues were fused in-frame at the 3′ end ofthe tub cDNA sequence. Site directed mutagenesis was employed asdescribed above, except that the primers utilized yielded fragmentscontaining the six histidine codons inserted just 5′ of the EcoRI siteat the 3′ end of the cDNA (i.e., CACCACCACCACCACCACTGAATTC); (SEQ IDNO:59). The resulting mutagenized fragment was excised and ligated intopET29* as described above to yield the murine pET29*-tub-HIS₆ expressionconstruct.

[0324] Human tub subcloning. The entire coding region of the human tubsequence was also inserted into the pET29* expression vector in bothnative and HIS₆ fusion forms. For insertion into pET29*, a human tubcDNA in pMal-C2 was modified via site directed mutagenesis to create 5′NdeI and 3′ EcoRI restriction sites, as described for the murine tubsequence, above, to yield the human pET29*-tub expression construct.

[0325] For construction of the human tub HIS₆ construct, six histidinecodons were introduced just 5′ of the EcoRI site by a three partligation. Specifically, a 5′ ApaLI-3′ EcoRI restriction fragmentencoding the last 25 amino acids of the murine pET29*-tub-HIS₆ wasexchanged for the equivalent fragment of the human tub gene sequence inhuman pET29*-tub construct, to yield the human pET29*-tub-HIS₆expression construct. It should be noted that, although the human andmouse tub genes have differing primary sequences, the amino acidresidues they encode within this carboxy-terminal region are identical.

[0326] Expression of recombinant tub proteins. Host bacteria BL21(DE3)(Novagen, Inc., Madison Wis.) were chemically transformed with each ofthe expression constructs described above (i.e., murine pET29*-tub,murine pET29*-tub-HIS₆, human pET29*-tub or human pET29*-tub-HIS₆) andgrown in 6 liters BHI (Brain Heart Infusion broth) cultures to mid-logphase (OD₅₉₅=1.0) at 37° C.

[0327] T7 RNA polymerase and, concomitantly, tub protein expression, wasinduced by the addition of IPTG to a final concentration of 0.5 mM. Twohours post-induction, bacteria were collected by centrifugation andfrozen.

[0328] Mammalian Expression

[0329] Murine tub subcloning. To prepare murine tub cDNA containing theentire tub coding region, the 5′ end of the murine tub cDNA in themurine pET29*-tub construct was mutagenized to remove the NdeIrestriction site, and replaced with a BamHI restriction site and a Kozakbox (Kozak, M. 1987, Nuc. Acid Res. 15:8125-8132) for efficientinitiation of translation in mammalian cells. After modification, thesequence just 5′ of the tub start codon was as follows: GGATCCACCATG(SEQ ID NO:60) (the start codon is underlined).

[0330] The modified sequence was digested with BamHI and EcoRI to excisethe region to be subcloned. After excision, the murine tub cDNA wasligated into the transient expression vector pN8ε (to yield the pN8ε-tubconstruct) and into the stable retroviral expression vector pWZLblast(to yield the pWZLblast-tub construct). Transcription in pN8ε isdirected from the human CMV immediate early promoter, whiletranscription from pWZLblast is initiated in the promoter embeddedMoloney Leukemia Virus LTR.

[0331] Constructs for the expression of epitope tagged recombinant tubgene product were generated, in which a DNA fragment encoding threetandem copies of the influenza virus hemagglutinin peptide YPYDVPDYA wasfused in-frame with the NH₂ terminus of the tub cDNA in pN8ε-tub.Specifically, the triple flu epitope was amplified from the plasmid pBSHA³ via PCR with primers possessing 5′ HindIII and 3′ BamHI restrictionsites. The PCR product was purified, HindIII/BamHI digested and ligatedinto HindIII/BamHI digested pN8ε-tub. The correct sequence of the fusionconstruct (designated pN8ε3Xflu-tub) was verified.

[0332] Expression of recombinant tub proteins. Transient expression isachieved by transfection of pN8ε-tub or pN8ε3Xflu-tub expressionconstructs into 293 EBNA cells (Invitrogen Corp.) via lipofection(Lipofectamine; Life Technologies Corp.). Analyses performed 72 hourspost-lipofection.

[0333] Stably infected polyclonal pools of NIH 3T3 cells harboringpWLZblast-tub proviruses are generated by transiently transfecting ΩEproducer cells (Morgenstern, J. P. & Land, H., 1990, Nuc. Acids Res.18:3587-3596) with calcium phosphate and harvesting recombinantretrovirus 48 hours later. The virus is then used to infect target NIH3T3 fibroblasts overnight at which time the infected cells are split1:10 into medium supplemented with blasticidin HCl (ICN Corp.) at aconcentration of 10 μg/ml. Colonies of blasticidinS HCl-resistant cellswhich appear within roughly two weeks are pooled and lysed for analysis.

15.2. Results

[0334] Described herein is the successful expression of recombinantmurine and human tub gene products in mammalian and/or bacterialsystems. With respect to bacterial expression, both native and HIS₆fusion (i.e., a fusion protein containing six carboxy-terminal histidineamino acid residues following the native tub amino acid sequence) tubgene products have been expressed. Details regarding the creation of tubexpression constructs and production of gene products using theseconstructs are described, above, in Section 15.1.

[0335] Aliquots of bacterial lysates (representing approximately 10⁻⁵ ofthe total 6 liter preparation were analyzed using standard SDSpolyacrylamide gel electrophoresis, as depicted in FIG. 11. A proteinwith a molecular weight of approximately 57 kD was readily apparent inproteins obtained from induced bacteria containing murine pET29*-tub. 57kD was the approximate molecular weight one would predict for the murinetub protein, with its 505 amino acid residues. Likewise, a protein witha molecular weight of approximately 57 kD was readily apparent inproteins obtained from induced bacteria containing human pET29*-tub. 57kD was the approximate molecular weight one would predict for the 506amino acid residue human tub gene product.

[0336] A protein exhibiting a slightly increased molecular weight wasreadily apparent in proteins obtained from induced bacteria containingeither human or murine pET29*-HIS₆. The slight increase in molecularweight was expected given the additional six histidine residues presentin these tub-HIS₆ fusion proteins.

[0337] Constructs for the expression of epitope-tagged murine tubprotein were utilized to demonstrate successful mammalian expression ofrecombinant tub gene product. Specifically, the pN8ε3Xflu-tub expressionconstruct was introduced, via lipofection, into 293 EBNA cells, asdescribed, above, in Section 15.1. After lipofection,immunoprecipitation followed by Western blot detection with themonoclonal antibody 12CA5 (directed against the flu hemagglutininpeptide; Boehringer Mannhein Corp.) was performed. Western blottingrevealed the presence of a protein exhibiting a molecular weight ofapproximately 59 kD (i.e., a size expected of the full length tub geneproduct fused the triple flu hemagglutinin peptide sequence). No suchprotein was detected in control transfections with non-hemagglutinintagged pN8ε-tub constructs.

[0338] In summary, the results described herein indicate thatrecombinant murine and human tub gene products have successfully beenexpressed in bacterial and/or mammalian systems.

16. EXAMPLE Identification and characterization of a tub Gene Homolog

[0339] The Example presented in this Section describes theidentification and characterization of a human tub gene homolog,referred to herein as human tub homolog 1.

[0340] The mouse tub gene nucleotide sequence was utilized as a databasequery using the Blastx program (1993, Nature Genetics 3:266-272), whichresulted in the identification of a human EST (GenBank Accession No.H92408) which exhibited a 75.3% identity over 85 derived amino acidresidues. The EST was originally derived from a human retinal library(Soares, B. and Benaldo, F.).

[0341] Upon identification of the EST, the corresponding clone wasobtained from Genbank and sequenced. A number of errors in the sequencelisted in the database were found. These included a deletion of bp 33from the Genbank sequence, incorrect base pair insertions (Genbanksequence bps 330, 339, 359, 366, 375 and 384), incorrect sequence at bps133-137 (ACCGA in Genbank sequence, as opposed to the correct GGCCGsequence) and incorrect bp 398 (T in Genbank as opposed to the correctG).

[0342] The identified sequence was used to screen a retinal cDNAlibrary, which resulted in the identification of several positiveclones. FIG. 12A-12C depicts nucleotide sequence of the tub homologidentified via this screening effort, which is referred to herein ashuman tub homolog 1. The sequence depicted in FIG. 12A-12C codes for asubstantial portion of the human tub homolog 1 protein, the derivedamino acid sequence of which is also depicted in FIG. 12A-12C. Thesequence exhibits a 73.9% identity over 216 derived amino acid residues.

[0343] The EST derived from the human tub homolog 1 gene was mapped inthe human by PCR typing of the Genebridge (G4) radiation hybridizationpanel. Typing of the DNA and comparison to radiation hybrid map data atthe Whitehead Institute Center for Genome Research (WICGR) tightlylinked the EST to an anonymous STS, WI-4186, on human chromosome 6.

[0344] Additionally, the EST was genetically mapped in the mouse using aC57BL/6J×Mus spretus interspecific backcross. Genotyping of 100 meiosesdemonstrated linkage to a region on mouse chromosome 17 between D17Mit48and D7Mit 9.

[0345] Human multiple tissue northern blots (Cat. No. 7766-1 and 7760-1;Clonetech, Palo Alto Calif.) containing 2 μg of poly A+ RNA per lanewere probed with the approximately 1.35 kb EcoRI-NotI fragment of thesequence obtained from Genbank containing the human tub homolog 1insert. The filters were prehybridized in 5 mls of Church buffer at 65°C. for 1 hour, after which 100 ng of ³²P-labelled probe was added. Probewas made using Stratagene Prime-It kit (Cat. No. 300392; Stratagene, LaJolla Calif.). Hybridization was allowed to proceed at 65° C. forapproximately 18 hours. Final washes of the filters was in 0.1% SDS,0.2×SSC solution for 65° C. Washed filters were exposed to aphosphoimager for 4 hours.

[0346] The Northern analysis was performed using a 1.35 kb probe asdescribed in Section 16.1, above, containing human tub homolog 1sequence encoding 285 amino acids plus 3′-untranslated sequence to thepoly-A sequence was performed. Tissues tested included brain, lung,liver, kidney, spleen, thymus, muscle, prostate, testis and fat. Amessage of approximately 2 kb was apparent in the lanes containing RNAfrom skeletal muscle and testis.

17. DEPOSIT OF MICROORGANISMS

[0347] The following microorganisms were deposited under the provisionsof the Budapest Treaty on the international Recognition of the Depositof Microorganisms for the Purposes of Patent Procedure, and comply withthe criteria set forth in 37 C.F.R. §1.801-1.809 regarding availabilityand permanency of deposits.

[0348] The following microorganisms were deposited with the AmericanType Culture Collection (ATCC),108001 University Boulevard, Manassas,Virginia 20110-2209, on the dates indicated and were assigned theindicated accession numbers: Microorganism Clone ATCC Access. No.Deposit Date H019 (E. coli) fumh019 69856 June 29, 1995 E/P8 (E. coli)P8 69858 June 30, 1995 E/P6 (E. coli) P6 69857 June 30, 1995 E/B13 (E.coli) B13 69859 June 30, 1995 CBT9H1 (E. coli) CBT9H1 97222 July 10,1995 CBT9H2 (E. coli) CBT9H2 97221 July 10, 1995 CBT9H3 (E. coli) CBT9H369874 July 28, 1995

[0349] The present invention is not to be limited in scope by thespecific embodiments described herein, which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed, various modifications of the invention, in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and accompanying drawings. Suchmodifications are intended to fall within the scope of the appendedclaims.

1 60 1804 base pairs nucleic acid single linear DNA CDS 139..1653 1CTGCAGGATT CGGCACGAGC AGCGGTCGGG CCGGGGAGGA TGCGGCCCGG GGCGGCCCGA 60GAGTTGAGCA GGGTCCCCGC GCCAGCCCCG AGCGGTCCCG GCCACCGGAG CCGCAGCCGC 120CGCCCCGCCC CCGGGAGA ATG ACT TCC AAG CCG CAT TCC GAC TGG ATT CCT 171 MetThr Ser Lys Pro His Ser Asp Trp Ile Pro 1 5 10 TAC AGT GTC CTA GAT GATGAG GGC AGC AAC CTG AGG CAG CAG AAG CTC 219 Tyr Ser Val Leu Asp Asp GluGly Ser Asn Leu Arg Gln Gln Lys Leu 15 20 25 GAC CGG CAG CGG GCC CTG TTGGAA CAG AAG CAG AAG AAG AAG CGC CAA 267 Asp Arg Gln Arg Ala Leu Leu GluGln Lys Gln Lys Lys Lys Arg Gln 30 35 40 GAG CCC TTG ATG GTA CAG GCC AATGCA GAT GGA CGG CCC CGG AGT CGG 315 Glu Pro Leu Met Val Gln Ala Asn AlaAsp Gly Arg Pro Arg Ser Arg 45 50 55 CGA GCC CGG CAG TCA GAG GAG CAA GCCCCC CTG GTG GAG TCC TAC CTC 363 Arg Ala Arg Gln Ser Glu Glu Gln Ala ProLeu Val Glu Ser Tyr Leu 60 65 70 75 AGC AGC AGT GGC AGC ACC AGC TAC CAAGTT CAA GAG GCC GAC TCG ATT 411 Ser Ser Ser Gly Ser Thr Ser Tyr Gln ValGln Glu Ala Asp Ser Ile 80 85 90 GCC AGT GTA CAG CTG GGA GCC ACC CGC CCACCA GCA CCA GCT TCA GCC 459 Ala Ser Val Gln Leu Gly Ala Thr Arg Pro ProAla Pro Ala Ser Ala 95 100 105 AAG AAA TCC AAG GGA GCG GCT GCA TCT GGGGGC CAG GGT GGA GCC CCT 507 Lys Lys Ser Lys Gly Ala Ala Ala Ser Gly GlyGln Gly Gly Ala Pro 110 115 120 AGG AAG GAG AAG AAG GGA AAG CAT AAA GGCACC AGC GGG CCA GCA ACT 555 Arg Lys Glu Lys Lys Gly Lys His Lys Gly ThrSer Gly Pro Ala Thr 125 130 135 CTG GCA GAA GAC AAG TCT GAG GCC CAA GGCCCA GTG CAG ATC TTG ACT 603 Leu Ala Glu Asp Lys Ser Glu Ala Gln Gly ProVal Gln Ile Leu Thr 140 145 150 155 GTG GGA CAG TCA GAC CAC GAC AAG GATGCG GGA GAG ACA GCA GCC GGC 651 Val Gly Gln Ser Asp His Asp Lys Asp AlaGly Glu Thr Ala Ala Gly 160 165 170 GGG GGC GCA CAG CCC AGT GGG CAG GACCTC CGT GCC ACG ATG CAG AGG 699 Gly Gly Ala Gln Pro Ser Gly Gln Asp LeuArg Ala Thr Met Gln Arg 175 180 185 AAG GGC ATC TCC AGC AGC ATG AGC TTTGAC GAG GAC GAG GAT GAG GAT 747 Lys Gly Ile Ser Ser Ser Met Ser Phe AspGlu Asp Glu Asp Glu Asp 190 195 200 GAA AAC AGC TCC AGC TCC TCC CAG CTAAAC AGC AAC ACC CGC CCT AGT 795 Glu Asn Ser Ser Ser Ser Ser Gln Leu AsnSer Asn Thr Arg Pro Ser 205 210 215 TCT GCC ACT AGC AGA AAG TCC ATC CGGGAG GCA GCT TCA GCC CCC AGC 843 Ser Ala Thr Ser Arg Lys Ser Ile Arg GluAla Ala Ser Ala Pro Ser 220 225 230 235 CCA GCC GCC CCA GAG CCA CCA GTGGAT ATT GAG GTC CAG GAT CTA GAG 891 Pro Ala Ala Pro Glu Pro Pro Val AspIle Glu Val Gln Asp Leu Glu 240 245 250 GAG TTT GCA CTG AGG CCA GCC CCACAA GGG ATC ACC ATC AAA TGC CGC 939 Glu Phe Ala Leu Arg Pro Ala Pro GlnGly Ile Thr Ile Lys Cys Arg 255 260 265 ATC ACT CGG GAC AAG AAG GGG ATGGAC CGC GGC ATG TAC CCC ACC TAC 987 Ile Thr Arg Asp Lys Lys Gly Met AspArg Gly Met Tyr Pro Thr Tyr 270 275 280 TTT CTG CAC CTA GAC CGT GAG GATGGC AAG AAG GTG TTC CTC CTG GCG 1035 Phe Leu His Leu Asp Arg Glu Asp GlyLys Lys Val Phe Leu Leu Ala 285 290 295 GGC AGG AAG AGA AAG AAG AGT AAAACT TCC AAT TAC CTC ATC TCT GTG 1083 Gly Arg Lys Arg Lys Lys Ser Lys ThrSer Asn Tyr Leu Ile Ser Val 300 305 310 315 GAC CCA ACA GAC TTG TCT CGGGGA GGC GAT AGC TAT ATC GGG AAA TTG 1131 Asp Pro Thr Asp Leu Ser Arg GlyGly Asp Ser Tyr Ile Gly Lys Leu 320 325 330 CGG TCC AAC CTG ATG GGC ACCAAG TTC ACC GTT TAT GAC AAT GGC GTC 1179 Arg Ser Asn Leu Met Gly Thr LysPhe Thr Val Tyr Asp Asn Gly Val 335 340 345 AAC CCT CAG AAG GCA TCC TCTTCC ACG CTG GAA AGC GGA ACC TTG CGC 1227 Asn Pro Gln Lys Ala Ser Ser SerThr Leu Glu Ser Gly Thr Leu Arg 350 355 360 CAG GAG CTG GCA GCG GTG TGCTAT GAG ACA AAT GTC CTA GGC TTC AAG 1275 Gln Glu Leu Ala Ala Val Cys TyrGlu Thr Asn Val Leu Gly Phe Lys 365 370 375 GGA CCT CGG AAG ATG AGT GTGATC GTC CCA GGC ATG AAC ATG GTT CAT 1323 Gly Pro Arg Lys Met Ser Val IleVal Pro Gly Met Asn Met Val His 380 385 390 395 GAG AGA GTC TGT ATC CGCCCC CGC AAT GAA CAT GAG ACC CTG TTA GCA 1371 Glu Arg Val Cys Ile Arg ProArg Asn Glu His Glu Thr Leu Leu Ala 400 405 410 CGC TGG CAG AAC AAG AACACG GAG AGC ATC ATT GAG CTG CAG AAC AAG 1419 Arg Trp Gln Asn Lys Asn ThrGlu Ser Ile Ile Glu Leu Gln Asn Lys 415 420 425 ACG CCA GTC TGG AAT GATGAC ACA CAG TCC TAT GTA CTT AAC TTC CAC 1467 Thr Pro Val Trp Asn Asp AspThr Gln Ser Tyr Val Leu Asn Phe His 430 435 440 GGC CGT GTC ACA CAG GCTTCT GTG AAG AAC TTC CAG ATC ATC CAC GGC 1515 Gly Arg Val Thr Gln Ala SerVal Lys Asn Phe Gln Ile Ile His Gly 445 450 455 AAT GAC CCG GAC TAC ATCGTC ATG CAG TTT GGC CGG GTA GCA GAA GAT 1563 Asn Asp Pro Asp Tyr Ile ValMet Gln Phe Gly Arg Val Ala Glu Asp 460 465 470 475 GTG TTC ACC ATG GATTAC AAC TAC CCA CTG TGT GCA CTG CAG GCC TTC 1611 Val Phe Thr Met Asp TyrAsn Tyr Pro Leu Cys Ala Leu Gln Ala Phe 480 485 490 GCC ATT GCT CTG TCCAGC TTT GAC AGC AAG CTG GCC TGC GAG 1653 Ala Ile Ala Leu Ser Ser Phe AspSer Lys Leu Ala Cys Glu 495 500 505 TAGAGGCCCC CCACTGCCGT TAGGTGGCCCAGTCCGGAGT GGAGCTTGCC TGCCTGCC 1713 GACAGGCCTG CCTACCCTCT GTTCATAGGCCCTCTATGGG CTTTCTGGTC TGACCAAC 1773 GAGATTGGTT TGCTCTGCCT CTGCTGCTTG A1804 505 amino acids amino acid unknown protein 2 Met Thr Ser Lys ProHis Ser Asp Trp Ile Pro Tyr Ser Val Leu Asp 1 5 10 15 Asp Glu Gly SerAsn Leu Arg Gln Gln Lys Leu Asp Arg Gln Arg Ala 20 25 30 Leu Leu Glu GlnLys Gln Lys Lys Lys Arg Gln Glu Pro Leu Met Val 35 40 45 Gln Ala Asn AlaAsp Gly Arg Pro Arg Ser Arg Arg Ala Arg Gln Ser 50 55 60 Glu Glu Gln AlaPro Leu Val Glu Ser Tyr Leu Ser Ser Ser Gly Ser 65 70 75 80 Thr Ser TyrGln Val Gln Glu Ala Asp Ser Ile Ala Ser Val Gln Leu 85 90 95 Gly Ala ThrArg Pro Pro Ala Pro Ala Ser Ala Lys Lys Ser Lys Gly 100 105 110 Ala AlaAla Ser Gly Gly Gln Gly Gly Ala Pro Arg Lys Glu Lys Lys 115 120 125 GlyLys His Lys Gly Thr Ser Gly Pro Ala Thr Leu Ala Glu Asp Lys 130 135 140Ser Glu Ala Gln Gly Pro Val Gln Ile Leu Thr Val Gly Gln Ser Asp 145 150155 160 His Asp Lys Asp Ala Gly Glu Thr Ala Ala Gly Gly Gly Ala Gln Pro165 170 175 Ser Gly Gln Asp Leu Arg Ala Thr Met Gln Arg Lys Gly Ile SerSer 180 185 190 Ser Met Ser Phe Asp Glu Asp Glu Asp Glu Asp Glu Asn SerSer Ser 195 200 205 Ser Ser Gln Leu Asn Ser Asn Thr Arg Pro Ser Ser AlaThr Ser Arg 210 215 220 Lys Ser Ile Arg Glu Ala Ala Ser Ala Pro Ser ProAla Ala Pro Glu 225 230 235 240 Pro Pro Val Asp Ile Glu Val Gln Asp LeuGlu Glu Phe Ala Leu Arg 245 250 255 Pro Ala Pro Gln Gly Ile Thr Ile LysCys Arg Ile Thr Arg Asp Lys 260 265 270 Lys Gly Met Asp Arg Gly Met TyrPro Thr Tyr Phe Leu His Leu Asp 275 280 285 Arg Glu Asp Gly Lys Lys ValPhe Leu Leu Ala Gly Arg Lys Arg Lys 290 295 300 Lys Ser Lys Thr Ser AsnTyr Leu Ile Ser Val Asp Pro Thr Asp Leu 305 310 315 320 Ser Arg Gly GlyAsp Ser Tyr Ile Gly Lys Leu Arg Ser Asn Leu Met 325 330 335 Gly Thr LysPhe Thr Val Tyr Asp Asn Gly Val Asn Pro Gln Lys Ala 340 345 350 Ser SerSer Thr Leu Glu Ser Gly Thr Leu Arg Gln Glu Leu Ala Ala 355 360 365 ValCys Tyr Glu Thr Asn Val Leu Gly Phe Lys Gly Pro Arg Lys Met 370 375 380Ser Val Ile Val Pro Gly Met Asn Met Val His Glu Arg Val Cys Ile 385 390395 400 Arg Pro Arg Asn Glu His Glu Thr Leu Leu Ala Arg Trp Gln Asn Lys405 410 415 Asn Thr Glu Ser Ile Ile Glu Leu Gln Asn Lys Thr Pro Val TrpAsn 420 425 430 Asp Asp Thr Gln Ser Tyr Val Leu Asn Phe His Gly Arg ValThr Gln 435 440 445 Ala Ser Val Lys Asn Phe Gln Ile Ile His Gly Asn AspPro Asp Tyr 450 455 460 Ile Val Met Gln Phe Gly Arg Val Ala Glu Asp ValPhe Thr Met Asp 465 470 475 480 Tyr Asn Tyr Pro Leu Cys Ala Leu Gln AlaPhe Ala Ile Ala Leu Ser 485 490 495 Ser Phe Asp Ser Lys Leu Ala Cys Glu500 505 437 base pairs nucleic acid single linear DNA 3 ACGGCAATGACCTTGAGTGT TGCCACTCCC TGTTTTTGAT GTTGTACGCA TGGTGCCCAG 60 CCCCCACCCCACCCCCAATC CCCTGATCTG GTCCATATCA GCCAGTGATG GGATGTGGGT 120 ATATGGCTTTTGTTAGAACT TTCTAACTGT AGTGATCTAG AGTCCTGCCC CTAGTGCCCT 180 GCATGTCTGGGGCTTGGGAA TACCCTTTAA ATGGATGTCT TTTCTCTCCT GGGCCCTGCT 240 GTCTGTGTGCATCTCCCCCC TTCACCCTCT TGCTTCATAA TGTTTCTCTT GAACCTTTGT 300 TTTCTTCATCCTTTCGATCT CTTTGGCATT TCTGCTTTCT CCTTCCCTCT TGTGGCCCAT 360 GTCTTACCTGGTCTCCCTGT CTCCACCATT CTTGCTTGTG CATTCCACAG CGGACTACAT 420 CGTCATGCATTTTGGCC 437 437 base pairs nucleic acid single linear DNA 4 ACGGCAATGACCGTGAGTGT TGCCACTCCC TGTTTTTGAT GTTGTACGCA TGGTGCCCAG 60 CCCCCACCCCACCCCCAATC CCCTGATCTG GTCCATATCA GCCAGTGATG GGATGTGGGT 120 ATATGGCTTTTGTAAGAACT TTCTAACTGT AGTGATCTAG AGTCCTGCCC CTAGTGCCCT 180 GCATGTCTGGGGCTTGGGAA TACCCTTTAA ATGGATGTCT TTTCTCTCCT GGGCCCTGCT 240 GTCTGTGTGCATCTCCCCCC TTCACCCTCT TGCTTCATAA TGTTTCTCTT GAACCTTTGT 300 TTTGTTCATCCTTTCGATCT CTTTGGCATT TCTGCTTTCT CCTTCCCTCT TGTGGCCCAT 360 GTCTTACCTGGTCTCCCTGT CTCCACCATT CTTGCTTGTG CATTCCACAG CGGACTACAT 420 CGTCATGCAGTTTGGCC 437 437 base pairs nucleic acid single linear DNA 5 ACGGCAATGACCTTGAGTGT TGCCACTCCC TGTTTTTGAT GTTGTACGCA TGGTGCCCAG 60 CCCCCACCCCACCCCCAATC CCCTGATCTG CTCCATATCA GCCAGTGATG GGATGTGGGT 120 ATATGGCTTTTGTTAGAACT TTCTAACTGT AGTGATCTAG AGTCCTGCCC CTAGTGCCCT 180 GCATGTCTGGGGCTTGGGAA TACCCTTTAA ATGGATGTCT TTTCTCTCCT GGGCCCTGCT 240 GTCTGTGTGCATCTCCCCCC TTCACCCTCT TGCTTCATAA TGTTTCTCTT GAACCTTTGT 300 TTTGTTCATCCTTTCGATCT CTTTGGCATT TCTGCTTTCT CCTTCCCTCT TGTGGCCCAT 360 GTCTTACCTGGTCTCCCTGT CTCCACCATT CTTGCTTGTG CATTCCACAG CGGACTACAT 420 CGTCATGCAGTTTGGCC 437 39 base pairs nucleic acid single linear DNA 6 ACGGCAATGACCCGGACTAC ATCGTCATGC AGTTTGGCC 39 2040 base pairs nucleic acid singlelinear DNA CDS 153..1670 7 TGGCGTGCAG CAGGGGCCTC GGCGGGGCCC AGCCCNCCGGTCCCGGGGAG GATACGTCCC 60 GGGGGCGGCC CGGGAGCTGA GCAGGCCCCC CGCGCCGGCCCCTCCGGGCC CCGGCCTCCA 120 GAGCCGCAGC CACCGCCCCG CCCCCGAGAG AC ATG ACTTCC AAG CCG CAT TCC 173 Met Thr Ser Lys Pro His Ser 1 5 GAC TGG ATT CCCTAC AGT GTC TTA GAT GAT GAG GGC AGA AAC CTG AGG 221 Asp Trp Ile Pro TyrSer Val Leu Asp Asp Glu Gly Arg Asn Leu Arg 10 15 20 CAG CAG AAG CTT GATCGG CAG CGG GCC CTG CTG GAG CAG AAG CAG AAG 269 Gln Gln Lys Leu Asp ArgGln Arg Ala Leu Leu Glu Gln Lys Gln Lys 25 30 35 AAG AAG CGC CAG GAG CCCCTG ATG GTG CAG GCC AAT GCA GAT GGG CGG 317 Lys Lys Arg Gln Glu Pro LeuMet Val Gln Ala Asn Ala Asp Gly Arg 40 45 50 55 CCC CGG AGC CGG CGG GCCCGG CAG TCA GAG GAA CAA GCC CCC CTG GTG 365 Pro Arg Ser Arg Arg Ala ArgGln Ser Glu Glu Gln Ala Pro Leu Val 60 65 70 GAG TCC TAC CTC AGC AGC AGTGGC AGC ACC AGC TAC CAA GTT CAA GAG 413 Glu Ser Tyr Leu Ser Ser Ser GlySer Thr Ser Tyr Gln Val Gln Glu 75 80 85 GCC GAC TCA CTC GCC AGT GTG CAGCTG GGA GCC ACG CGC CCA ACA GCA 461 Ala Asp Ser Leu Ala Ser Val Gln LeuGly Ala Thr Arg Pro Thr Ala 90 95 100 CCA GCT TCA GCC AAG AGA ACC AAGGCG GCA GCT ACA GCA GGG GGC CAG 509 Pro Ala Ser Ala Lys Arg Thr Lys AlaAla Ala Thr Ala Gly Gly Gln 105 110 115 GGT GGC GCC GCT AGG AAG GAG AAGAAG GGA AAG CAC AAA GGC ACC AGC 557 Gly Gly Ala Ala Arg Lys Glu Lys LysGly Lys His Lys Gly Thr Ser 120 125 130 135 GGG CCA GCA GCA CTG GCA GAAGAC AAG TCT GAG GCC CAA GGC CCA GTG 605 Gly Pro Ala Ala Leu Ala Glu AspLys Ser Glu Ala Gln Gly Pro Val 140 145 150 CAG ATT CTG ACT GTG GGC CAGTCA GAC CAC GCC CAG GAC GCA GGG GAG 653 Gln Ile Leu Thr Val Gly Gln SerAsp His Ala Gln Asp Ala Gly Glu 155 160 165 ACG GCA GCT GGT GGG GGC GAACGG CCC AGC GGG CAG GAT CTC CGT GCC 701 Thr Ala Ala Gly Gly Gly Glu ArgPro Ser Gly Gln Asp Leu Arg Ala 170 175 180 ACG ATG CAG AGG AAG GGC ATCTCC AGC AGC ATG AGC TTT GAC GAG GAT 749 Thr Met Gln Arg Lys Gly Ile SerSer Ser Met Ser Phe Asp Glu Asp 185 190 195 GAG GAG GAT GAG GAG GAG AATAGC TCC AGC TCC TCC CAG CTA AAT AGT 797 Glu Glu Asp Glu Glu Glu Asn SerSer Ser Ser Ser Gln Leu Asn Ser 200 205 210 215 AAC ACC CGC CCC AGC TCTGCT ACT AGC AGG AAG TCC GTC AGG GAG GCA 845 Asn Thr Arg Pro Ser Ser AlaThr Ser Arg Lys Ser Val Arg Glu Ala 220 225 230 GCC TCA GCC CCT AGC CCAACA GCT CCA GAG CAA CCA GTG GAC GTT GAG 893 Ala Ser Ala Pro Ser Pro ThrAla Pro Glu Gln Pro Val Asp Val Glu 235 240 245 GTC CAG GAT CTT GAG GAGTTT GCA CTG AGG CCG GCC CCC CAG GGT ATC 941 Val Gln Asp Leu Glu Glu PheAla Leu Arg Pro Ala Pro Gln Gly Ile 250 255 260 ACC ATC AAA TGC CGC ATCACT CGG GAC AAG AAA GGG ATG GAC CGG GGC 989 Thr Ile Lys Cys Arg Ile ThrArg Asp Lys Lys Gly Met Asp Arg Gly 265 270 275 ATG TAC CCC ACC TAC TTTCTG CAC CTG GAC CGT GAG GAT GGG AAG AAG 1037 Met Tyr Pro Thr Tyr Phe LeuHis Leu Asp Arg Glu Asp Gly Lys Lys 280 285 290 295 GTG TTC CTC CTG GCGGGA AGG AAG AGA AAG AAG AGT AAA ACT TCC AAT 1085 Val Phe Leu Leu Ala GlyArg Lys Arg Lys Lys Ser Lys Thr Ser Asn 300 305 310 TAC CTC ATC TCT GTGGAC CCA ACA GAC TTG TCT CGA GGA GGG GAC AGC 1133 Tyr Leu Ile Ser Val AspPro Thr Asp Leu Ser Arg Gly Gly Asp Ser 315 320 325 TAT ATC GGG AAA CTGCGG TCC AAC TTG ATG GGC ACC AAG TTC ACT GTT 1181 Tyr Ile Gly Lys Leu ArgSer Asn Leu Met Gly Thr Lys Phe Thr Val 330 335 340 TAT GAC AAT GGA GTCAAC CCT CAG AAG GCC TCA TCC TCC ACT TTG GAA 1229 Tyr Asp Asn Gly Val AsnPro Gln Lys Ala Ser Ser Ser Thr Leu Glu 345 350 355 AGT GGA ACC TTA CGTCAG GAG CTG GCA GCT GTG TGC TAC GAG ACA AAC 1277 Ser Gly Thr Leu Arg GlnGlu Leu Ala Ala Val Cys Tyr Glu Thr Asn 360 365 370 375 GTC TTA GGC TTCAAG GGG CCT CGG AAG ATG AGC GTG ATT GTC CCA GGC 1325 Val Leu Gly Phe LysGly Pro Arg Lys Met Ser Val Ile Val Pro Gly 380 385 390 ATG AAC ATG GTTCAT GAG AGA GTC TCT ATC CGC CCC CGC AAC GAG CAT 1373 Met Asn Met Val HisGlu Arg Val Ser Ile Arg Pro Arg Asn Glu His 395 400 405 GAG ACA CTG CTAGCA CGC TGG CAG AAT AAG AAC ACG GAG AGT ATC ATC 1421 Glu Thr Leu Leu AlaArg Trp Gln Asn Lys Asn Thr Glu Ser Ile Ile 410 415 420 GAG CTG CAA AACAAG ACA CCT GTC TGG AAT GAT GAC ACA CAG TCC TAT 1469 Glu Leu Gln Asn LysThr Pro Val Trp Asn Asp Asp Thr Gln Ser Tyr 425 430 435 GTA CTC AAC TTCCAT GGG CGC GTC ACA CAG GCC TCC GTG AAG AAC TTC 1517 Val Leu Asn Phe HisGly Arg Val Thr Gln Ala Ser Val Lys Asn Phe 440 445 450 455 CAG ATC ATCCAT GGC AAT GAC CCG GAC TAC ATC GTG ATG CAG TTT GGC 1565 Gln Ile Ile HisGly Asn Asp Pro Asp Tyr Ile Val Met Gln Phe Gly 460 465 470 CGG GTA GCAGAG GAT GTG TTC ACC ATG GAT TAC AAC TAC CCG CTG TGT 1613 Arg Val Ala GluAsp Val Phe Thr Met Asp Tyr Asn Tyr Pro Leu Cys 475 480 485 GCA CTG CAGGCC TTT GCC ATT GCC CTG TCC AGC TTC GAC AGC AAG CTG 1661 Ala Leu Gln AlaPhe Ala Ile Ala Leu Ser Ser Phe Asp Ser Lys Leu 490 495 500 GCG TGC GAGTAGAGGCCTC TTCGTGCCCT TTGGGGTTGC CCAGCCTGGA 1710 Ala Cys Glu 505GCGGAGCTTG CCTGCCTGCC TGTGGAGACA GCCCTGCCTA TCCTCTGTAT ATAGGCCTTC 1770CGCCAGATGA AGCTTTGGCC CTCAGTGGGC TCCCTGGCCC AGCCAGCCAG GAACTGGCTC 1830CTTTGGCTCT GCTACTGAGG CAGGGGAGTA GTGGAGAGCG GGTGGGTGGG TGTTGAAGGG 1890ATTGAGAATT AATTCTTTCC ATGCCACGAG GATCAACACA CACTCCCACC CTTGGGTAGT 1950AAGTGGTTGT TGTNAGTCGG TACTTTACCA AAGCTTGAGC AACCTCTTCC AAGCTTGGGA 2010AAGGGCCGCA AAAAGGCATT AGGAGGGGAG 2040 506 amino acids amino acid unknownprotein 8 Met Thr Ser Lys Pro His Ser Asp Trp Ile Pro Tyr Ser Val LeuAsp 1 5 10 15 Asp Glu Gly Arg Asn Leu Arg Gln Gln Lys Leu Asp Arg GlnArg Ala 20 25 30 Leu Leu Glu Gln Lys Gln Lys Lys Lys Arg Gln Glu Pro LeuMet Val 35 40 45 Gln Ala Asn Ala Asp Gly Arg Pro Arg Ser Arg Arg Ala ArgGln Ser 50 55 60 Glu Glu Gln Ala Pro Leu Val Glu Ser Tyr Leu Ser Ser SerGly Ser 65 70 75 80 Thr Ser Tyr Gln Val Gln Glu Ala Asp Ser Leu Ala SerVal Gln Leu 85 90 95 Gly Ala Thr Arg Pro Thr Ala Pro Ala Ser Ala Lys ArgThr Lys Ala 100 105 110 Ala Ala Thr Ala Gly Gly Gln Gly Gly Ala Ala ArgLys Glu Lys Lys 115 120 125 Gly Lys His Lys Gly Thr Ser Gly Pro Ala AlaLeu Ala Glu Asp Lys 130 135 140 Ser Glu Ala Gln Gly Pro Val Gln Ile LeuThr Val Gly Gln Ser Asp 145 150 155 160 His Ala Gln Asp Ala Gly Glu ThrAla Ala Gly Gly Gly Glu Arg Pro 165 170 175 Ser Gly Gln Asp Leu Arg AlaThr Met Gln Arg Lys Gly Ile Ser Ser 180 185 190 Ser Met Ser Phe Asp GluAsp Glu Glu Asp Glu Glu Glu Asn Ser Ser 195 200 205 Ser Ser Ser Gln LeuAsn Ser Asn Thr Arg Pro Ser Ser Ala Thr Ser 210 215 220 Arg Lys Ser ValArg Glu Ala Ala Ser Ala Pro Ser Pro Thr Ala Pro 225 230 235 240 Glu GlnPro Val Asp Val Glu Val Gln Asp Leu Glu Glu Phe Ala Leu 245 250 255 ArgPro Ala Pro Gln Gly Ile Thr Ile Lys Cys Arg Ile Thr Arg Asp 260 265 270Lys Lys Gly Met Asp Arg Gly Met Tyr Pro Thr Tyr Phe Leu His Leu 275 280285 Asp Arg Glu Asp Gly Lys Lys Val Phe Leu Leu Ala Gly Arg Lys Arg 290295 300 Lys Lys Ser Lys Thr Ser Asn Tyr Leu Ile Ser Val Asp Pro Thr Asp305 310 315 320 Leu Ser Arg Gly Gly Asp Ser Tyr Ile Gly Lys Leu Arg SerAsn Leu 325 330 335 Met Gly Thr Lys Phe Thr Val Tyr Asp Asn Gly Val AsnPro Gln Lys 340 345 350 Ala Ser Ser Ser Thr Leu Glu Ser Gly Thr Leu ArgGln Glu Leu Ala 355 360 365 Ala Val Cys Tyr Glu Thr Asn Val Leu Gly PheLys Gly Pro Arg Lys 370 375 380 Met Ser Val Ile Val Pro Gly Met Asn MetVal His Glu Arg Val Ser 385 390 395 400 Ile Arg Pro Arg Asn Glu His GluThr Leu Leu Ala Arg Trp Gln Asn 405 410 415 Lys Asn Thr Glu Ser Ile IleGlu Leu Gln Asn Lys Thr Pro Val Trp 420 425 430 Asn Asp Asp Thr Gln SerTyr Val Leu Asn Phe His Gly Arg Val Thr 435 440 445 Gln Ala Ser Val LysAsn Phe Gln Ile Ile His Gly Asn Asp Pro Asp 450 455 460 Tyr Ile Val MetGln Phe Gly Arg Val Ala Glu Asp Val Phe Thr Met 465 470 475 480 Asp TyrAsn Tyr Pro Leu Cys Ala Leu Gln Ala Phe Ala Ile Ala Leu 485 490 495 SerSer Phe Asp Ser Lys Leu Ala Cys Glu 500 505 605 base pairs nucleic acidsingle linear DNA 9 AGCCTACAGT TTAAACAGTC GACTCTAGAC TTAATTAAGGNTCCGGNGCG CCCCCGGGTA 60 CCGAGCTCTG GTCTCACCCA CTGCCTGTTT CTCTCTCTCCATCTGGGGAT GTTTCCTGAG 120 CAGTTCAAGA GGCCGACTCA CTCGCCAGTG TGCAGCTGGGAGCCACGCGC CCAACAGCAC 180 CAGCTTCAGC CAAGAGAACC AAGGCGGCAG CTACAGCAGGGGGCCAGGGC GGCGCCGCTA 240 GGAAGGAGAA GAAGGGAAAG CACAAAGGTC AGCTCACATTCTCTACAGCC CTGCCCAGCA 300 GGCCTGGCCT CCACTGTAGG GCTGGGGAAG GTTTGTCCTCCTGACTTGGA GGGGAGGGAT 360 AGGATGAACA GCCTCAGGGA AGACACAGAC TGCCACTCTGGGCACCCCCT CAGGTGGCTC 420 ACAGGCCTCA TCTAGCTTGG GAGGTGCCTG GGCTGCCTCTGGGTGTGGGC ATGCCTACCA 480 ACACTGCCAG GAAGTGAAGT CCTGCTCAGC TTTGGCCCAGAACCACCGTC CCNANCTTNA 540 GTTACTTTGG CCTTGAGGAA CCTTTATNAT GACCCCNTNAAGGAGGATTT TAACCAAGCT 600 GGATT 605 826 base pairs nucleic acid singlelinear DNA 10 TTCAAGGGCC AAAGTTTTTT AATGATGTAT GGGAGTTAAT GAAGGNGGTATGTGGGTNTG 60 TTNGNGGAAG AAAACACCAG CATTGATGGT TGTAGNTGKT GGTGTCCAKGAATGATTGCT 120 GGCCTTGCCT ATGGTNTGGA TCAGTCCTTG TTNTCCCATC TTGTTTTTTCCCATGTGCAG 180 TTGGTTTTTG TAGATGGCTG CCGTCTGCTT TAAAGGACGT GAGGTGTTGTAAACCAACCC 240 TCGGCAATTA ATTTGGGGGA AGAGCAGAAG AAATGAAGCC CAACATCCCTTACTAGCTTA 300 CCAGTTGTTA ACAGGCTGGT GCAATCATTA GTTTTATAAA AATCAGTTTTGCAAATAAAG 360 TTTTGCAGAG GGTTTCCCCA CTCTTCCCTC ATCCCCTTCA TGGACGTCTGAGAATCCAGG 420 CCCTCCTCTC CTCCTCCTGG ATGTAACTCA GGCGTGTCCG TGGCCTGCAGGCACCAGCGG 480 GCCAGCAGCA CTGGCAGAAG ACAAGWCTGA GGCCCAAGGC CCAGTGCAGATTCTGACTGT 540 GGGCCAGTCA GACCACGCCC AGGACGCAGG GGAGACGGCA GCTGGTGGGGGCGAACGGCC 600 CAGCGGGCAG GATCTCCGTK CCACGATGCA GAGGAAGGGT GAGCCCCATGGGGGCCCAGT 660 GATACCCCCA AAACTCAGTC CCAGGTTCTC AGATGCACCT TTCTCTGGGAGCATGGNCTT 720 CCTGTGTCCA AACCCCTCCC TGGCAATGGT GGGTGAGGGT GGGGCACACTTCGGAGACAA 780 ATNAGAAACT CTTAGGCAGG GNCCCTGCTA AGGCCCCAGG GAGGCC 8261943 base pairs nucleic acid single linear DNA 11 TTAAACAGTC GACTCTAGACTTAATTAAGG ATCCGGCGCG CCCCCGGGTA CCGAGCTCAG 60 TGCAGGCCTT GATACACAAGAGACAGTGGT AGGGTGSCTG CTAGGTAGTG GGGTAATGTA 120 GGGACTGAGC TGAAACTGGGTGGTGGGGAT ATATCCTGAG GATTGTGGCC AGCCCCGGCT 180 CATGTGTGTA CCTGAGAGAATATCCTTTTA TATCTGGACA TGTGTGGGAA TATATGTGTG 240 AATGGGAGTC TATATGTGTAGATATGGCTA AGAGTGTGTG CATAAGTTTG TGGGGGTACA 300 GGTGAGTCAG TGTCTGAACATGAGTATGTG ACCATGTGTA TTTCAGGGGC AGGGTAGACT 360 TCTCCTCATT CATCCCTTCTTCTTCTCTCC TTGGCCCAGG CATCTCCAGC AGCATGAGCT 420 TTGACGAGGA TGAGGAGGATGAGGAGGAGA ATAGCTCCAG CTCCTCCCAG CTAAATAGTA 480 ACACCCGCCC CAGCTCTGCTACTAGCAGGA AGTCCGTCAG GGTGAGTGAG TGAGTCTGCA 540 TCCACAGCAG TTTTTGGAGGACTGCTCATC CGTTAGAGGT GGACTGCATG TGAAGAGATG 600 GACTCGTATG CCTTTAGGAGCTTCTCTGCT GGCCTCTTAC GTCCCTCTAC CTTGCCTCCT 660 AACCTCTTCA GCTAGGCCAGCAGGGTGATG TATGGGGGGA GATGCAGTTG GACAGGATGA 720 CCTCTGAGGA CCTCCCGTATCTCCCATCTC CACCTCTAGG AACTGTTGAG GGCAGGGCTG 780 GGAAGATAGC TTCTGACCCCAGGCCCAGGC TGGCCAGGCC CCAATCCCAG GATCCTTCCC 840 TCTCTCCCAC CGCCACGTTAGGAGGCAGAT TTGGATCCCA GACCACCAAT TTGGGCTGCT 900 TAGGGTCCTT GGGGCTCAGGCACCTATTCT GCATCCCCAT AGGAGGCAGC CTCAGCCCCT 960 AGCCCAACAG CTCCAGAGCAACCAGTGGAC GTTGAGGTCC AGGATCTTGA GGAGTTTGCA 1020 CTGAGGCCGC CCCCCCAGGGTATCACCATC AAATGCCGCA TCACTCGGGA CAAGAAAGGG 1080 ATGGACCGGG GCATGTACCCCACCTACTTT CTGCACCTGG ACCGTGAGGA TGGGAAGAAG 1140 GTAAGGTTGG TCTGGGCATGTTATCATCTA GGCTTTACAG CCCTTTGAAA TCCTAGGGGC 1200 TGAAATGTGA CTGGAAGTCTCATATCTACC GCTGACCTCT CAGTTCCTCA AAGAAACTGC 1260 CTTCGTGTCT GGTCTGTGCACATCTTTGTG TTTTCCAGTG CATTTGTGTG TGTGCACATA 1320 TGTGCGTTTG GGAGCTGACGCAACGGAGAG AGTCTGTGTG AGTGGCTCTC ATGACTGTGT 1380 GCAGACCAGA GGCTGAGTCTGGAATATGAC CTCATTCCAC TCCCCAAGGT GTTCCTCCTG 1440 GCGGGAAGGA AGAGAAAGAAGAGTAAAACT TCCAATTACC TCATCTCTGT GGACCCAACA 1500 GACTTGTCTC GAGGAGGGGACAGCTATATC GGGAAACTGC GGGTACTAGC ATTCCCCCAG 1560 GAAGCAGGCG GGAGTGGGAGGGAGGGGCAG GGGCAAGCTG TCTGTAGAGG GCCTGAATCT 1620 TCCTGAAGGA GATCTAGGCCAGGGATGGAT ACTCTCCCAG GATCCTCTCT GATAATCACA 1680 TCCAACTGGA GGCCTATGTCTATGCCAGCC TAGAGCCAGA CTTGGAGATG GGACTCACAC 1740 ACCCGACCCC AAGCTGTTCCCAGGAGGTGG GTGCAGGCCC ACCAAGAGTG ATGGATCCAA 1800 CCCCAGGGTG TCACTGATAACGCAGGCCAC CATGGAAGAG TTGCCTTGGC TCCATGGTCA 1860 ATGCCAAGGG ACAGGGCTGAGAGTGAGCTC GGTACCCGGG GGCGCKCCGG ATCCTTAATT 1920 AAGTCTAGAG TCGACTGTTTAAG 1943 881 base pairs nucleic acid single linear DNA 12 GATTTAGNGGAACACAGCAN CTTGNGGGTG GGANGGCAGT GGTGAAGGGG CAGGAAGGCT 60 CTGAGCCTAGGCCTCCAGGT GGGGGCAGTG GGGAGGTAGG GTTTGCTGAG GAACTGAGTA 120 CCAGATTTGGGGAGCATAAA TAAAGATGAG AGGTCAGGAG CTAAAGCTGG AGATGGGGCT 180 GGACTGAGACTTAGGCTGGC TGCGACAGAG GAGATCTCAT CCTCTCTCCA CGGGTGCTAA 240 GCCTCTTCCACTGTCTTATC AGATGCCATT CTGTTTGCTC ACCTCCCATG AGGAGAACTC 300 CCATGTTCCCCCAGATAAAT CTYCTGAAGA ATCCTGATTG ACCTCCCTGA ATTGCTCTCA 360 CTGAACTGAAATGCACTTTG AGTCAACTCA GAGCAAGTCC AGGCCTTCTG CCCACGAAGT 420 GTCTTCAAAGATGTGGATTC AGTGAGCAGT ATGCCTCCCT GGGCCTGCTC CTGTTCCAGC 480 CCAGAATGTTTTGCAGGCTC CTCATAGGAC AGACGATGAG CTGTTCCCTG CTTCTGGGGC 540 AGAGGGTGCATGACTCTATA CTGATTGTGC CTTTATTTCA GGTCCAACTT GATGGGCACC 600 AAGTTCACTGTTTATGACAA TGGAGTCAAC CCTCAGAAGG CCTCATCCTC CACTTTGGAA 660 AGTGGAACCTTACGTCAGGA GCTGGCAGCT GTGTGCTACG TGAGTCCTAG GTTCGGGGGT 720 CTCTGATTTCCAAGGTAGAT ATGAAATCCA GGACTTGATG CCTGATCTAG GGGCTATCCC 780 ATCCATCTTAGTGGGTAGAC AAGGCTGTGT GGAGAGGGGC TGTCCTCTGT GGAGTGTTCC 840 TGGCCTAGGACAGGGGCTCT GGCTCTCTCC TCCTGACTTC A 881 1622 base pairs nucleic acidsingle linear DNA 13 AGTAGTTTGC CGGAYCGAAG TGGAAGAACA RCATTCCCGTGAGCAGAACC AAGGATGACG 60 CATAAGAGGA GCTAGTTCTG GCAGGGTAGA GACCCCAGGGGCTCAGTTCT GGCCCGTGTT 120 AGGTTTAGAG GGATGTGTGT TAGACTTCGG AGTGGAGATGGTGGGAACTA GCTCTTCCTC 180 TTTATTCCCG TCCCCCCCAC CTTCTCCAGT AGGTAAATAGACGCCTCAGG TGGCCAGTGT 240 TGCGTTCTCT TTCCCAGGAG ACAAACGTCT TAGGCTTCAAGGGVCCTCGG AAGATGAGCG 300 TGATTGTCCC AGGCATGAAC ATGGTTCATG AGAGAGTCTCTATCCGCCCC CGCAACGTGA 360 GTGTCTACCC CTTCCTCCCC TCTTTCCCCA TCATCCTAGTCTCTGCATGA GCTTCTAAGG 420 GCAGAACTCC AGCTGATGTG TATATGTGGA GGGGTACCATGTGAGAAAGC CCTGGAGGTC 480 TAGGGAAATC CAAGGACCCC CATTCCCGGG ATAGATCCCTTTCTGGGGTG GTCATGGTGC 540 CAAAGGCCTG GGCCTGGCTC AGGTGAGGCT GCCCTCCCAGGAGCATGAGA CACTGCTAGC 600 ACGCTGGCAG AATAAGAACA CGGAGTGTAT CATCGAGCTGCAAAACAAGA CACCTGTCTG 660 GAATGATGAC ACACAGTCCT ATGTACTCAA CTTCCATGGGCGCGTCACAC AGGCCTCCGT 720 GAAGAACTTC CAGATCATCC ATGGCAATGA CCGTGAGTGTTTCTGTCCCT ACTCATTATG 780 GTCCGTAGGA TACCCAAGGC CCTTAGCGTA GGGTTCAGCCCACCTAGCCC TGCCTACACT 840 GGCTAGAGTT TAAGAATGTG AGCTATACAG CTAAGGTTAGATGTATGGAA CTTTCTAACC 900 CTAATGACTG GGAGGTCCTG GAAGAACCTT CTTTGSAGCCCTGGTCCTAG ATTCTGTGTA 960 TTCAACGGAG TCTCAGGCAC GGGAACACCC TTTAAAAGGACTTTTCCTCT TTTCTGTCCC 1020 CTGGTGTTCA CATGCATCTT ACTTTGTCCT TTGSCATCTGCCACCTCTTT CCTGCCACTT 1080 CTCCCAATTG GCCTTTGTTT TACTTCCCTT TGTGATTCCCCTGGCATCTC TGCTTCTCAC 1140 TTGTTCTTCC CTCATGTGGT TTGGGTGTCT GTCTATCCTTCCCTGGCTCT ACCATTCCTG 1200 TCCTGTCCTT TTCTCTGTCT GTGCCTGTGC TTGGCCCCAGCGGACTACAT CGTGATGCAG 1260 TTTGGCCGGG TAGCAGAGGA TGTGTTCACC ATGGATTACAACTACCCGCT GTGTGCACTG 1320 CAGGCCTTTG CCATTGCCCT GTCCAGCTTC GACAGCAAGCTGGCGTGCGA GTAGAGGCCT 1380 CTTCGTGCCC TTTGGGGTTG CCCAGCCTGG AGCGGAGCTTGCCTGCCTGC CTGTGGAGAC 1440 AGCCCTGCCT ATCCTCTGTA TATAGGCCTT CCGCCAGATGAAGCTTTGGC CCTCAGTGGG 1500 CTCCCTGGCC CAGCCAGCCA GGAACTGGCT CCTTTGCCTCTGCTACTGAG GCAGGGGAGT 1560 AGTGGAGAGC GGGTGGGTGG GTGTGAAGGG ATGAGAATAATTCTTTCCAT GCCACGAGAT 1620 CC 1622 1338 base pairs nucleic acid singlelinear DNA CDS 1..855 14 GTG ATA AAG AAC AGC AAT CAA AAG GGC AAA GCC AAAGGA AAA GGC AAA 48 Val Ile Lys Asn Ser Asn Gln Lys Gly Lys Ala Lys GlyLys Gly Lys 1 5 10 15 AAG AAA GCG AAG GAG GAG AGG GCC CCG TCT CCC CCCGTG GAG GTG GAC 96 Lys Lys Ala Lys Glu Glu Arg Ala Pro Ser Pro Pro ValGlu Val Asp 20 25 30 GAA CCC CGG GAG TTT GTG CTC CGG CCT GCC CCC CAG GGCCGC ACG GTG 144 Glu Pro Arg Glu Phe Val Leu Arg Pro Ala Pro Gln Gly ArgThr Val 35 40 45 CGC TGC CGG CTG ACC CGG GAC AAA AAG GGC ATG GAT CGA GGCATG TAT 192 Arg Cys Arg Leu Thr Arg Asp Lys Lys Gly Met Asp Arg Gly MetTyr 50 55 60 CCC TCC TAC TTC CTG CAC CTG GAC ACG GAG AAG AAG GTG TTC CTCTTG 240 Pro Ser Tyr Phe Leu His Leu Asp Thr Glu Lys Lys Val Phe Leu Leu65 70 75 80 GCT GGC AGG AAA CGA AAA CGG AGC AAG ACA GCC AAT TAC CTC ATCTCC 288 Ala Gly Arg Lys Arg Lys Arg Ser Lys Thr Ala Asn Tyr Leu Ile Ser85 90 95 ATC GAC CCT ACC AAT CTG TCC CGA GGA GGG GAG AAT TTC ATC GGG AAG336 Ile Asp Pro Thr Asn Leu Ser Arg Gly Gly Glu Asn Phe Ile Gly Lys 100105 110 CTG AGG TCC AAC CTC CTG GGG AAC CGC TTC ACG GTC TTT GAC AAC GGG384 Leu Arg Ser Asn Leu Leu Gly Asn Arg Phe Thr Val Phe Asp Asn Gly 115120 125 CAG AAC CCA CAG CGT GGG TAC AGC ACT AAT GTG GCA AGC CTT CGG CAG432 Gln Asn Pro Gln Arg Gly Tyr Ser Thr Asn Val Ala Ser Leu Arg Gln 130135 140 GAG CTG GCA GCT GTG ATC TAT GAA ACC AAC GTG CTG GGC TTC CGT GGC480 Glu Leu Ala Ala Val Ile Tyr Glu Thr Asn Val Leu Gly Phe Arg Gly 145150 155 160 CCC CGG CGC ATG ACC GTC ATC ATT CCT GGC ATG AGT GCG GAG AACGAG 528 Pro Arg Arg Met Thr Val Ile Ile Pro Gly Met Ser Ala Glu Asn Glu165 170 175 AGG GTC CCC ATC CGG CCC CGA AAT GCT AGT GAC GGC CTG CTG GTGCGC 576 Arg Val Pro Ile Arg Pro Arg Asn Ala Ser Asp Gly Leu Leu Val Arg180 185 190 TGG CAG AAC AAG ACG CTG GAG AGC CTC ATA GAA CTG CAC AAC AAGCCA 624 Trp Gln Asn Lys Thr Leu Glu Ser Leu Ile Glu Leu His Asn Lys Pro195 200 205 CCT GTC TGG AAC GAT GAC AGT GGC TCC TAC ACC CTC AAC TTC CAAGGC 672 Pro Val Trp Asn Asp Asp Ser Gly Ser Tyr Thr Leu Asn Phe Gln Gly210 215 220 CGG GTC ACC CAG GCC TCA GTC AAG AAC TTC CAG ATT GTC CAC GCTGAT 720 Arg Val Thr Gln Ala Ser Val Lys Asn Phe Gln Ile Val His Ala Asp225 230 235 240 GAC CCC GAC TAT ATC GTG CTG CAG TTC GGC CGC GTG GCG GAGGAC GCC 768 Asp Pro Asp Tyr Ile Val Leu Gln Phe Gly Arg Val Ala Glu AspAla 245 250 255 TTC ACC CTA GAC TAC CGG TAC CCG CTG TGC GCC CTG CAG GCCTTC GCC 816 Phe Thr Leu Asp Tyr Arg Tyr Pro Leu Cys Ala Leu Gln Ala PheAla 260 265 270 ATC GCC CTC TCC AGT TTC GAC GGG AAG CTG GCC TGC GAGTGACCCCAGC 865 Ile Ala Leu Ser Ser Phe Asp Gly Lys Leu Ala Cys Glu 275280 285 AGCCCCTCAG CGCCCCCAGA GCCCGTCAGC GTGGGGGAAA GGATTCAGTGGAGGCTGGCA 925 GGGTCCCTCC AGCAAAGCTC CCGCGGAAAA CTGCTCCTGT GTCGGGGCTGACCTCTCACT 985 GCCTCTCGGT GACCTCCGTC CTCTCCCCAG CCTGGCACAG GCCGAGGCAGGAGGAGCCCG 1045 GACGGCGGGT AGGACGGAGA TGAAGAACAT CTGGAGTTGG AGCCGCACATCTGGTCTCGG 1105 AGCTCGCCTG CGCCGCTGTG CCCCCCTCCT CCCCGCGCCC CAGTCACTTCCTGTCCGGGA 1165 GCAGTAGTCA TTGTTGTTTT AACCTCCCCT CTCCCCGGGA CCGCGCTAGGGCTCCGAGGA 1225 GCTGGGGCGG GCTAGGAGGA GGGGGTAGGT GATGGGGGAC GAGGGCCAGGCACCCACATC 1285 CCCAATAAAG CCGCGTCCTT GGCAAAAAAA AAAAAAAAAA AAAAAAAAAAAAA 1338 285 amino acids amino acid unknown protein 15 Val Ile Lys AsnSer Asn Gln Lys Gly Lys Ala Lys Gly Lys Gly Lys 1 5 10 15 Lys Lys AlaLys Glu Glu Arg Ala Pro Ser Pro Pro Val Glu Val Asp 20 25 30 Glu Pro ArgGlu Phe Val Leu Arg Pro Ala Pro Gln Gly Arg Thr Val 35 40 45 Arg Cys ArgLeu Thr Arg Asp Lys Lys Gly Met Asp Arg Gly Met Tyr 50 55 60 Pro Ser TyrPhe Leu His Leu Asp Thr Glu Lys Lys Val Phe Leu Leu 65 70 75 80 Ala GlyArg Lys Arg Lys Arg Ser Lys Thr Ala Asn Tyr Leu Ile Ser 85 90 95 Ile AspPro Thr Asn Leu Ser Arg Gly Gly Glu Asn Phe Ile Gly Lys 100 105 110 LeuArg Ser Asn Leu Leu Gly Asn Arg Phe Thr Val Phe Asp Asn Gly 115 120 125Gln Asn Pro Gln Arg Gly Tyr Ser Thr Asn Val Ala Ser Leu Arg Gln 130 135140 Glu Leu Ala Ala Val Ile Tyr Glu Thr Asn Val Leu Gly Phe Arg Gly 145150 155 160 Pro Arg Arg Met Thr Val Ile Ile Pro Gly Met Ser Ala Glu AsnGlu 165 170 175 Arg Val Pro Ile Arg Pro Arg Asn Ala Ser Asp Gly Leu LeuVal Arg 180 185 190 Trp Gln Asn Lys Thr Leu Glu Ser Leu Ile Glu Leu HisAsn Lys Pro 195 200 205 Pro Val Trp Asn Asp Asp Ser Gly Ser Tyr Thr LeuAsn Phe Gln Gly 210 215 220 Arg Val Thr Gln Ala Ser Val Lys Asn Phe GlnIle Val His Ala Asp 225 230 235 240 Asp Pro Asp Tyr Ile Val Leu Gln PheGly Arg Val Ala Glu Asp Ala 245 250 255 Phe Thr Leu Asp Tyr Arg Tyr ProLeu Cys Ala Leu Gln Ala Phe Ala 260 265 270 Ile Ala Leu Ser Ser Phe AspGly Lys Leu Ala Cys Glu 275 280 285 20 base pairs nucleic acid singlelinear DNA 16 CCGACTCGAT TGCCAGTGTA 20 20 base pairs nucleic acid singlelinear DNA 17 GCGGATACAG ACTCTCTCAT 20 21 base pairs nucleic acid singlelinear DNA 18 GTTCAAGCTG GTTTCAAGAT G 21 20 base pairs nucleic acidsingle linear DNA 19 ATCATCCAGG GAAGATGGAC 20 19 base pairs nucleic acidsingle linear DNA 20 CTTCCTGGTG GAGGCAGTG 19 20 base pairs nucleic acidsingle linear DNA 21 GAAGCAGTGA CGGGATGTGG 20 18 base pairs nucleic acidsingle linear DNA 22 GGGTACCGAG CTCTGGTC 18 20 base pairs nucleic acidsingle linear DNA 23 TCCAAGTCAG GAGGACAAAC 20 21 base pairs nucleic acidsingle linear DNA 24 GAAAGTGCAT CTGAGAACCT G 21 20 base pairs nucleicacid single linear DNA 25 CCTCCTCCTG GATGTAACTC 20 21 base pairs nucleicacid single linear DNA 26 TGTGACCATG TGTATTTCAG G 21 20 base pairsnucleic acid single linear DNA 27 CCTCTAACGG ATGAGCAGTC 20 20 base pairsnucleic acid single linear DNA 28 GATTTGGATC CCAGACCACC 20 21 base pairsnucleic acid single linear DNA 29 GACTTCCAGT CACATTTCAG C 21 18 basepairs nucleic acid single linear DNA 30 GTGCAGACCA GAGGCTGA 18 20 basepairs nucleic acid single linear DNA 31 TTCAGGCCCT CTACAGACAG 20 20 basepairs nucleic acid single linear DNA 32 TCATAGGACA GACGATGAGC 20 21 basepairs nucleic acid single linear DNA 33 GTCCTGGATT TCATATCTAC C 21 20base pairs nucleic acid single linear DNA 34 AGGTAAATAG ACGCCTCAGG 20 20base pairs nucleic acid single linear DNA 35 ACGTCTGCCC TTAGAAGCTC 20 18base pairs nucleic acid single linear DNA 36 CTGGACCTGG CTCAGGTG 18 22base pairs nucleic acid single linear DNA 37 GTCATTAGGG TTAGAAAGTT CC 2220 base pairs nucleic acid single linear DNA 38 TCTTCCCTCA TGTGGTTTGG 2019 base pairs nucleic acid single linear DNA 39 CCACAGGCAG GCAGGCAAG 1920 base pairs nucleic acid single linear DNA 40 TGCGCAGAAA CAATCACCTA 2017 base pairs nucleic acid single linear DNA 41 CAAGACGTGA ACCTGGA 17 20base pairs nucleic acid single linear DNA 42 GCGGATACAG ACTCTCTCAT 20 20base pairs nucleic acid single linear DNA 43 GAGGACAAAT GTCCTAGGCT 20 17base pairs nucleic acid single linear DNA 44 CATGCTCCTT GGGATGT 17 17base pairs nucleic acid single linear DNA 45 TGAGGATTGC TTAAAGA 17 90base pairs nucleic acid single linear DNA 46 GAGACAAATG TCCTAGGCTTCAAGGGACCT CGGAAGATGA GTGTGATCGT CCCAGGCATG 60 AACATGGTTC ATGAGAGAGTCTGTATCCGC 90 19 base pairs nucleic acid single linear DNA 47 GGACAAGAAGGGGATGGAC 19 19 base pairs nucleic acid single linear DNA 48 CCGTGGATGATCTGGAAGT 19 20 base pairs nucleic acid single linear DNA 49 TGAGACAAATGTCCTAGGCT 20 20 base pairs nucleic acid single linear DNA 50 TGGACAGAGCAATGGCGAAG 20 20 base pairs nucleic acid single linear DNA 51 CCGACTCGATTGCCAGTGTA 20 20 base pairs nucleic acid single linear DNA 52 GCGGATACAGACTCTCTCAT 20 20 base pairs nucleic acid single linear DNA 53 CCGACTCGATTGCCAGTGTA 20 21 base pairs nucleic acid single linear DNA 54 GGAGCTGTTTTCATCCTCAT C 21 20 base pairs nucleic acid single linear DNA 55GAAGGAGAAG AAGGGAAAGC 20 20 base pairs nucleic acid single linear DNA 56GGGTGTTACT ATTTAGCTGG 20 20 base pairs nucleic acid single linear DNA 57TTCAAGAGGC CGACTCGATT 20 19 base pairs nucleic acid single linear DNA 58TTCCTCTGCA TCGTGGCAC 19 25 base pairs nucleic acid single linear DNA 59CACCACCACC ACCACCACTG AATTC 25 12 base pairs nucleic acid single linearDNA 60 GGATCCACCA TG 12

What is claimed is:
 1. A method for identifying a compound useful formodulating tub gene expression, comprising: (a) contacting a testcompound with a cell or cell lysate containing a tub gene nucleic acidmolecule; and (b) measuring the level of expression of the tub genenucleic acid molecule in the cell, such that if the level obtained in(b) differs from that obtained in the absence of the test compound, thena compound that modulates tub gene expression is identified.
 2. Themethod of claim 1 wherein the level of expression of the tub genenucleic acid molecule is assayed by determining a change in body-weightphenotype.
 3. The method of claim 1 wherein the level of expression ofthe tub gene nucleic acid molecule is assayed by measuring the level oftub protein.
 4. The method of claim 1 wherein the level of expression ofthe tub gene nucleic acid molecule is assayed by measuring mRNAtranscripts of the tub gene.
 5. A method for identifying a compounduseful for modulating tub gene expression, comprising: (a) contacting atest compound with a cell or cell lysate containing mRNA transcripts ofthe tub gene; and (b) detecting the level of translation of the tub mRNAtranscripts, such that if the level obtained in (b) differs from thatobtained in the absence of the test compound, then a compound thatmodulates tub gene expression is identified.
 6. The method of claim 5wherein the level of translation of the tub mRNA transcripts is detectedby measuring the level of tub protein.
 7. A method for identifying acompound useful for modulating tub gene expression, comprising: (a)contacting a test compound with a cell or cell lysate containing a tubgene nucleic acid molecule operatively associated with a reporter gene;and (b) detecting the level of expression of the reporter gene, suchthat if the level obtained in (b) differs from that obtained in theabsence of the test compound, then a compound that modulates tub geneexpression is identified.
 8. A method for detecting a compound usefulfor the treatment of a body weight disorder comprising the method ofclaim 1, 5, or 7, further comprising the step of administering the testcompound to a non-human animal, and determining whether the testcompound alters the body weight of the treated animal.
 9. The method ofclaim 1, 5, or 7 wherein the test compound increases tub geneexpression.
 10. The method of claim 1, 5, or 7 wherein the cell or celllysate is a recombinant cell or cell lysate.
 11. The method of claim 1,5, or 7 wherein the cell is a non-recombinant cell or cell lysate. 12.13. The method of claim 1, 5 or 7 wherein the cell is of a cell linethat expresses the tub gene.
 14. The method of claim 13 wherein the cellline is a hypothalamic cell line.
 15. The method of claim 14 wherein thehypothalamic cell line is a GH-1 or GN hypothalamic cell line.
 16. Themethod of claim 1, 5 or 7 wherein the test compound comprises a smallorganic molecule, small inorganic molecule, a soluble peptide, or anantibody.